Systemic immune activation method using nucleic acid-lipid complexes

ABSTRACT

This invention relates to a method for systemic immune activation which is effective for eliciting both a systemic, non-antigen specific immune response and a strong antigen-specific immune response in a mammal. The method is particularly effective for protecting a mammal from a disease including cancer, a disease associated with allergic inflammation, or an infectious disease. Also disclosed are therapeutic compositions useful in such a method.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional application of U.S. patentapplication Ser. No. 09/104,759, filed Jun. 25, 1998, which isspecifically incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to a composition and method toelicit an immune response in a mammal using a genetic immunizationstrategy. More particularly, the present invention includes compositionsand methods for eliciting systemic, non-specific (i.e.,non-antigen-specific) immune responses in a mammal as well asantigen-specific immune responses, both of which are useful inimmunization protocols.

BACKGROUND OF THE INVENTION

[0003] Vaccines are widely used to prevent disease and to treatestablished diseases (therapeutic vaccines). There remains, however, anurgent need to develop safe and effective vaccines and adjuvants for avariety of diseases, including those due to infection by pathogenicagents, cancers and other disorders amenable to treatment by elicitationof an immune response.

[0004] Three major types of disease in mammals which are amenable toelicitation and/or modulation of an immune response include infectiousdiseases, allergic inflammatory diseases and cancer, although thepresent invention is not limited to treatment of these disease types.Infectious diseases are caused by infectious agents (i.e., infectiousdisease pathogens), examples of which include viruses, bacteria,parasites, yeast and other fungi. In allergic inflammatory diseases,allergens cause the release of inflammatory mediators that recruit cellsinvolved in inflammation in allergic or sensitized animals, the presenceof which can lead to tissue damage and sometimes death. Cancer canresult from an inherited inability to repair DNA, to prevent DNA damageor to prevent propagation of cells with damaged DNA, and/or from abiochemical dysfunction or genetic mutation which leads to uncontrolledcell proliferation and DNA synthesis.

[0005] Traditional reagents that are used in an attempt to protect amammal from such diseases include reagents that destroy infectiousagents or the cells involved in deregulated biological functions, orthat modify the activity of such cells. Such reagents, however, canresult in unwanted side effects. For example, anti-viral drugs thatdisrupt the replication of viral DNA also often disrupt DNA replicationin normal cells in the treated patient. The use of anti-inflammatory andsymptomatic relief reagents in allergic inflammation is a seriousproblem because of their side effects or their failure to attack theunderlying cause of an inflammatory response. Other treatments withchemotherapeutic reagents to destroy cancer cells typically leads toside effects, such as bleeding, vomiting, diarrhea, ulcers, hair lossand increased susceptibility to secondary cancers and infections.

[0006] An alternative method of disease treatment includes modulatingthe immune system of a patient to assist the patient's natural defensemechanisms. Traditional reagents and methods used to attempt to regulatean immune response in a patient also result in unwanted side effects andhave limited effectiveness. For example, immunopharmacological reagentsused to treat cancer (e.g., interleukins) are short-lived in thecirculation of a patient and are ineffective except in large doses. Dueto the medical importance of immune regulation and the inadequacies ofexisting immunopharmacological reagents, reagents and methods toregulate specific parts of the immune system have been the subject ofstudy for many years.

[0007] Vaccines can be used not only to prevent disease, but can also beused to treat established diseases (i.e., therapeutic vaccines). Anumber of tumor antigens that are recognized by T lymphocytes of theimmune system have been recently identified and are being considered aspotential vaccine candidates. Conventional vaccines generally consist ofeither (1) purified antigens administered with an adjuvant, or (2) anattenuated form of a pathogen that can be administered to a patient togenerate an immune response, but not cause serious disease or illness.

[0008] Genetic vaccines, by contrast, contain a DNA sequence thatencodes an antigen(s) against which the immune response is to begenerated. For genetic vaccines to generate an antigen-specific immuneresponse, the gene of interest must be expressed in the mammalian host.Gene expression has been accomplished by use of viral vectors (e.g.,adenovirus, poxvirus) that express the foreign gene of interest in thevaccinated patient and induce an immune response against the encodedprotein. Alternatively, plasmid DNA encoding a foreign gene has beenused to induce an immune response. The primary routes of administrationof these so-called “naked” DNA vaccines are intramuscular orpercutaneous. It is generally accepted that viral vector systems inducebetter immune responses than naked DNA systems, probably because theviral delivery systems induce more inflammation and immune activationthan naked DNA vaccines. The propensity of viral vaccines to inducenon-specific immune responses, primarily as a result of viral componentrecognition by the complement cascade, also represents a potentialdrawback, however, since such immune responses often preventreadministration of the vaccine.

[0009] Therefore, there is need to provide better vaccines which canproduce an immune response which is safe, antigen-specific and effectiveto prevent and/or treat diseases amenable to treatment by elicitation ofan immune response, such as infectious disease, allergy and cancer.

SUMMARY

[0010] One embodiment of the present invention generally relates to amethod to elicit a systemic, non-antigen-specific immune response in amammal. The method includes the step of administering to the mammal atherapeutic composition by a route of administration selected fromintravenous and intraperitoneal administration. The therapeuticcomposition includes: (a) a liposome delivery vehicle; and, (b) anisolated nucleic acid molecule that is not operatively linked to atranscription control sequence. In another embodiment, the route ofadministration is intravenous. In further embodiments of the method, theisolated nucleic acid molecule comprises a non-coding sequence. In oneembodiment, the isolated nucleic acid molecule does not comprise abacterial nucleic acid sequence.

[0011] Accordingly, another embodiment of the present invention is acomposition for eliciting a systemic, non-antigen-specific immuneresponse in a mammal. Such a composition includes (a) a liposomedelivery vehicle; and (b) an isolated nucleic acid molecule that is notoperatively linked to a transcription control sequence. In oneembodiment, the nucleic acid molecule does not include a bacterialnucleic acid sequence.

[0012] Another embodiment of the present invention relates to acomposition for eliciting a systemic, non-antigen-specific immuneresponse in a mammal which comprises (a) a liposome delivery vehicle and(b) an isolated non-coding nucleic acid sequence.

[0013] A composition of the present invention can further comprise apharmaceutically acceptable excipient. A pharmaceutically acceptableexcipient can include, for example a non-ionic diluent, and morepreferably, 5 percent dextrose in water (D5W).

[0014] The above-mentioned method and compositions of the presentinvention have the advantages of eliciting a systemic, non-antigenspecific immune response in a mammal, and more particularly, ofeliciting a systemic, anti-viral immune response in a mammal.Additionally, the method and composition of the present invention canelicit a systemic, anti-tumor immune response in a mammal. Such ananti-tumor immune response can result in the reduction of a tumor in themammal. The method and composition of the present invention can alsoelicit a systemic, protective immune response against allergicinflammation in a mammal. The systemic, non-antigen-specific immuneresponse elicited by the method and composition of the present inventionresult in an increase in effector cell activity, and particularly,natural killer (NK) cell activity in the mammal, and additionally canresult in increased production of IFN* in the mammal.

[0015] Yet another embodiment of the present invention relates to amethod to elicit an immunogen-specific immune response and a systemic,non-specific immune response in a mammal. The method includesadministering to the mammal a therapeutic composition by a route ofadministration selected from intravenous and intraperitoneal. Thetherapeutic composition comprises: (a) a liposome delivery vehicle; and,(b) a recombinant nucleic acid molecule comprising an isolated nucleicacid sequence encoding an immunogen, wherein the nucleic acid sequenceis operatively linked to a transcription control sequence. Particularlysuitable transcription control sequences include Rous sarcoma virus(RSV) control sequences, cytomegalovirus (CMV) control sequences,adenovirus control sequences and Simian virus (SV-40) control sequences.This method of the present invention has the particular advantage ofeliciting both a systemic, non-immunogen-specific immune response in amammal, as well as an immunogen-specific immune response that have apotent therapeutic effect in the mammal. In one embodiment, the route ofadministration is intravenous. In other preferred embodiments, theimmunogen is a tumor antigen, an infectious disease pathogen antigen oran allergen.

[0016] When the mammal has cancer, this immunogen is preferably a tumorantigen. In one embodiment of this method, the therapeutic compositioncan include a plurality of recombinant nucleic acid molecules, each ofthe recombinant nucleic acid molecules comprising a cDNA sequenceamplified from total RNA isolated from an autologous tumor sample, eachof the cDNA sequences encoding a tumor antigen or a fragment thereof andbeing operatively linked to a transcription control sequence. In anotherembodiment, the therapeutic composition comprises a plurality ofrecombinant nucleic acid molecules, each of the recombinant nucleic acidmolecules comprising a cDNA sequence amplified from total RNA isolatedfrom a plurality of allogeneic tumor samples of the same histologicaltumor type, each of the cDNA sequences encoding a tumor antigen or afragment thereof and being operatively linked to a transcription controlsequence.

[0017] The methods and compositions of the present invention areparticularly useful for treating a cancer which includes melanomas,squamous cell carcinoma, breast cancers, head and neck carcinomas,thyroid carcinomas, soft tissue sarcomas, bone sarcomas, testicularcancers, prostatic cancers, ovarian cancers, bladder cancers, skincancers, brain cancers, angiosarcomas, hemangiosarcomas, mast celltumors, primary hepatic cancers, lung cancers, pancreatic cancers,gastrointestinal cancers, renal cell carcinomas, hematopoieticneoplasias, and metastatic cancers thereof. The compositions and methodsof the present invention are especially useful for treating primary lungcancer or pulmonary metastatic cancer.

[0018] Accordingly, a tumor antigen useful in the present composition ispreferably from a cancer selected from the group of melanomas, squamouscell carcinoma, breast cancers, head and neck carcinomas, thyroidcarcinomas, soft tissue sarcomas, bone sarcomas, testicular cancers,prostatic cancers, ovarian cancers, bladder cancers, skin cancers, braincancers, angiosarcomas, hemangiosarcomas, mast cell tumors, primaryhepatic cancers, lung cancers, pancreatic cancers, gastrointestinalcancers, renal cell carcinomas, hematopoietic neoplasias and metastaticcancers thereof. The tumor antigen preferably is selected from the groupof tumor antigens having epitopes that are recognized by T cells, tumorantigens having epitopes that are recognized by B cells, tumor antigensthat are exclusively expressed by tumor cells, and/or tumor antigensthat are expressed by tumor cells and by non-tumor cells.

[0019] When the immunogen is a tumor antigen which is expressed in themammal, the method of the present invention produces a result selectedfrom alleviation of the cancer, reduction of size of a tumor associatedwith the cancer, elimination of a tumor associated with the cancer,prevention of metastatic cancer, prevention of the cancer andstimulation of effector cell immunity against the cancer. When the tumorantigen is administered intravenously, the antigen is expressed in apulmonary tissue of the mammal and prevents pulmonary metastatic cancerin the mammal.

[0020] When the immunogen is an infectious disease pathogen antigen, themethods and composition of the present invention are useful for mammalshaving an infectious disease, and particularly for mammals having achronic infectious disease. Such immunogens can be from infectiousdisease pathogens which include bacteria, viruses, parasites and fungi.Such infectious disease pathogens include, for example, humanimmunodeficiency virus (HIV), Mycobacterium tuberculosis, herpesvirus,papillomavirus and Candida. The present method is particularly usefulwhen the infectious disease pathogen is a virus, and more particularly,human immunodeficiency virus and feline immunodeficiency virus. Inanother embodiment, the present method is particularly useful when theinfectious disease is tuberculosis. In this embodiment, the immunogencan be, for example, a Mycobacterium tuberculosis antigen, or morespecifically, antigen 85.

[0021] Expression of the pathogen antigen in a tissue of the mammalproduces a result selected from the group of alleviation of the disease,regression of established lesions associated with the disease,alleviation of symptoms of the disease, immunization against the diseaseand/or stimulation of effector cell immunity against the disease.

[0022] In one embodiment of this method, the therapeutic compositioncomprises a plurality of recombinant nucleic acid molecules, each of therecombinant nucleic acid molecules comprising a cDNA sequence amplifiedfrom total RNA isolated from an infectious disease pathogen, each of thecDNA sequences encoding an immunogen from the infectious diseasepathogen or a fragment thereof and being operatively linked to atranscription control sequence.

[0023] When the mammal has a disease associated with allergicinflammation, the immunogen is an allergen. Suitable allergens include,plant pollens, drugs, foods, venoms, insect excretions, molds, animalfluids, animal hair and animal dander. This method is particularlyuseful when the mammal has a disease selected from allergic airwaydiseases, allergic rhinitis, allergic conjunctivitis, and food allergy.Expression of the allergen in a tissue of the mammal produces a resultselected from the group consisting of alleviation of the disease,alleviation of symptoms of the disease, desensitization against thedisease, and stimulation of a protective immune response against thedisease.

[0024] In another embodiment of this method, the therapeutic compositioncomprises a plurality of recombinant nucleic acid molecules, each of therecombinant nucleic acid molecules comprising a cDNA sequence amplifiedfrom total RNA isolated from an allergen, each of the cDNA sequencesencoding the allergen or a fragment thereof and being operatively linkedto a transcription control sequence.

[0025] Yet another embodiment of the present invention relates to amethod to elicit a systemic, non-specific immune response in a mammal,which includes administering to the mammal a therapeutic composition bya route of administration selected from intravenous and intraperitoneal,wherein the therapeutic composition comprises: (a) a liposome deliveryvehicle; and, (b) a recombinant nucleic acid molecule comprising anisolated nucleic acid sequence encoding a cytokine, the nucleic acidsequence being operatively linked to a transcription control sequence.The method of the present invention is particularly useful for elicitinga systemic, anti-viral immune response or a systemic; an anti-tumorimmune response; a systemic, protective immune response against allergicinflammation in the mammal; and/or for reduction of a tumor in themammal. Additionally, the method increases production of IFN* in themammal and/or increases natural killer (NK) cell activity in the mammal.In one embodiment, the route of administration is intravenous. Thecytokine can include hematopoietic growth factors, interleukins,interferons, immunoglobulin superfamily molecules, tumor necrosis factorfamily molecules and/or chemokines. In one embodiment, the cytokine isan interleukin, and in a more preferred embodiment, the interleukin isselected from the group of interleukin-2 (IL-2), interleukin-7 (IL-7),interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18)or interferon-* (IFN*), and in an even more preferred embodiment, theinterleukin is selected from the group of interleukin-2 (IL-2),interleukin-12 (IL-12), interleukin-18 (IL-18) or interferon-* (IFN*).

[0026] Another embodiment of the present invention relates to a methodto elicit a tumor antigen-specific immune response and a systemic,non-specific immune response in a mammal that has cancer. The methodincludes administering to a mammal a therapeutic composition by a routeof administration selected from intravenous and intraperitonealadministration. The therapeutic composition comprises: (a) a liposomedelivery vehicle; and, (b) total RNA isolated from a tumor sample, theRNA encoding tumor antigens. In one embodiment, the route ofadministration is intravenous. In another embodiment, the RNA isenriched for poly-A RNA prior to administration to the mammal.

[0027] Yet another embodiment of the present invention relates to amethod to elicit a pathogen-antigen-specific immune response and asystemic, non-specific immune response in a mammal that has aninfectious disease. Such method includes administering to a mammal atherapeutic composition by a route of administration selected fromintravenous and intraperitoneal administration, the therapeuticcomposition comprising: (a) a liposome delivery vehicle; and, (b) totalRNA isolated from an infectious disease pathogen, the RNA encodingpathogen antigens. In another embodiment, the route of administration isintravenous.

[0028] Another embodiment of the present invention relates to acomposition for systemic administration to a mammal to elicit animmunogen-specific immune response and a systemic, non-specific immuneresponse. The composition includes (a) a liposome delivery vehicle; and(b) a recombinant nucleic acid molecule comprising an isolated nucleicacid sequence encoding an immunogen, the nucleic acid sequence beingoperatively linked to a transcription control sequence. The compositionhas a nucleic acid:lipid ratio of from about 1:1 to about 1:64.

[0029] In one embodiment, any of the above compositions of the presentinvention administered to a mammal by the present methods can include arecombinant nucleic acid molecule having a nucleic acid sequenceencoding a cytokine. In this embodiment, the nucleic acid sequenceencoding a cytokine is operatively linked to a transcription controlsequence. In the compositions which include a nucleic acid sequenceencoding an immunogen, the nucleic acid sequence encoding a cytokine canbe in the same or separate recombinant nucleic acid molecule whichcontains the nucleic acid sequence encoding the immunogen. The nucleicacid sequence encoding a cytokine and the nucleic acid sequence encodingan immunogen can be operatively linked to the same or differenttranscription control sequences. In preferred embodiments, the cytokineis selected from the group of hematopoietic growth factors,interleukins, interferons, immunoglobulin superfamily molecules, tumornecrosis factor family molecules and/or chemokines. In one embodiment,the cytokine is an interleukin, and in a more preferred embodiment, theinterleukin is selected from the group of interleukin-2 (IL-2),interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15),interleukin-18 (IL-18) or interferon-* (IFN*), and in an even morepreferred embodiment, the interleukin is selected from the group ofinterleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-18 (IL-18) orinterferon-* (IFN*).

[0030] Liposome delivery vehicles suitable for use in any of thecompositions and methods of the present invention can include anyliposomes. Particularly preferred liposomes are cationic liposomes.Other preferred liposomes include multilamellar vesicle lipids andextruded lipids, with multilamellar vesicle lipids being more preferred.Liposome compositions can include, but are not limited to, pairs oflipids selected from DOTMA and cholesterol, DOTAP and cholesterol, DOTIMand cholesterol, and DDAB and cholesterol, with DOTAP and cholesterolbeing particularly preferred.

[0031] The compositions of the present invention administered by thepresent methods have a nucleic acid:lipid ratio of from about 1:1 toabout 1:64. In some embodiments, the compositions have a nucleicacid:lipid ratio of from about 1:10 to about 1:40. Other suitable ratiosare additionally set forth below.

[0032] The methods and compositions of the present invention arepreferably used to elicit an immune response in a mammal, which includeshumans, dogs, cats, mice, rats, sheep, cattle, horses or pigs, and morepreferably, humans.

[0033] Additional advantages and novel features of this invention shallbe set forth in part in the description that follows, and in part willbecome apparent to those skilled in the art upon examination of thefollowing specification or may be learned by the practice of theinvention. The advantages of the invention may be realized and attainedby means of the instrumentalities, combinations, compositions, andmethods particularly pointed out in the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

[0034]FIG. 1 is a bar graph illustrating that intravenous injection ofCLDC induces marked activation of 5 different immune effectorpopulations in vivo.

[0035]FIG. 2A is a bar graph showing that intravenous injection of CLDC,but not lipid or DNA alone, induces immune activation of CD8+ cells invivo.

[0036]FIG. 2B is a bar graph showing that intravenous injection of CLDC,but not lipid or DNA alone, induces immune activation of NK1.1+ cells invivo.

[0037]FIG. 3 is a bar graph comparing the immune activating potencies ofLPS, poly I/C and CLDC in vivo.

[0038]FIG. 4 is a bar graph is a bar graph showing in vivo doseresponses for immune activation by CLDC.

[0039]FIG. 5 is a bar graph illustrating the influence of route ofadministration of CLDC on immune activation.

[0040]FIG. 6 is a bar graph showing that immune activation can beinduced by CLDC formed with several different lipids.

[0041]FIG. 7 is a bar graph demonstrating that immune activation by CLDCis independent of the DNA source.

[0042]FIG. 8 is a bar graph illustrating that IFN* release by immunecells is induced by administration of CLDC, but not lipid or DNA alone.

[0043]FIG. 9 is a bar graph showing that administration of CLDC, but notpoly I/C or LPS, induces IFN* production by splenocytes in vivo.

[0044]FIG. 10A is a bar graph showing that NK cells are the source ofIFN* production in splenocytes elicited by intravenous administration ofCLDC injection.

[0045]FIG. 10B is a bar graph showing that NK cells are the source ofIFN* production in lung mononuclear cells elicited by intravenousadministration of CLDC injection.

[0046]FIG. 11 is a line graph illustrating that administration of CLDCinduces high levels of NK activity in splenocytes.

[0047]FIG. 12A is a bar graph showing that intraperitonealadministration of CLDC induces immune activation in CD8+ splenocytes invivo.

[0048]FIG. 12B is a bar graph showing that intraperitonealadministration of CLDC induces immune activation in NK1.1+splenocytes invivo.

[0049]FIG. 13A is a bar graph demonstrating that CLDC exert potentantitumor effects against fibrosarcoma tumor cells in vivo.

[0050]FIG. 13B is a bar graph demonstrating that CLDC exert potentantitumor effects against melanoma tumor cells in vivo.

[0051]FIG. 13C is a bar graph demonstrating that CLDC exert potentantitumor effects against colon carcinoma tumor cells in vivo.

[0052]FIG. 13D is a bar graph demonstrating that CLDC exert potentantitumor effects against breast cancer tumor cells in vivo.

[0053]FIG. 14 is a bar graph showing that systemic administration ofCLDC, but not DNA or lipid alone, induces antitumor activity in vivo.

[0054]FIG. 15 is a bar graph demonstrating that the antitumor activityof CLDC is independent of the DNA source.

[0055]FIG. 16 is a bar graph showing that the type of CLDC administeredsignificantly influences antitumor activity.

[0056]FIG. 17A is a bar graph illustrating that intravenousadministration of CLDC induces selective gene expression in pulmonarytissues.

[0057]FIG. 17B is a bar graph illustrating that intravenousadministration of CLDC encoding IL-2 induces intrapulmonary IL-2expression.

[0058]FIG. 17C is a bar graph illustrating that intravenousadministration of CLDC encoding IFN* induces intrapulmonary IFN*expression.

[0059]FIG. 18A is a bar graph showing that day 3 administration of CLDCencoding 3 different cytokine genes improves the antitumor activityagainst fibrosarcoma tumor cells in vivo over empty vector alone.

[0060]FIG. 18B is a bar graph showing that day 3 administration of CLDCencoding 3 different cytokine genes improves the antitumor activityagainst colon carcinoma tumor cells in vivo over empty vector alone.

[0061]FIG. 18C is a bar graph showing that day 3 administration of CLDCencoding 3 different cytokine genes improves the antitumor activityagainst melanoma tumor cells in vivo over empty vector alone.

[0062]FIG. 18D is a bar graph showing that day 6 administration of CLDCencoding 3 different cytokine genes improves the antitumor activityagainst fibrosarcoma tumor cells in vivo over empty vector alone.

[0063]FIG. 18E is a bar graph showing that day 6 administration of CLDCencoding 3 different cytokine genes improves the antitumor activityagainst colon carcinoma tumor cells in vivo over empty vector alone.

[0064]FIG. 18F is a bar graph showing that day 6 administration of CLDCencoding 3 different cytokine genes improves the antitumor activityagainst melanoma tumor cells in vivo over empty vector alone.

[0065]FIG. 19A is a line graph illustrating that intravenousadministration of CLDC encoding ovalbumin induces strong, systemic,antigen-specific immune responses in vivo.

[0066]FIG. 19B is a line graph demonstrating that intravenousimmunization with CLDC encoding an antigen is at least 10 times morepotent immune inducer of immune activation than intramuscular injectionof DNA encoding an antigen.

[0067]FIG. 20 is a bar graph showing that systemic immunization withCLDC encoding a tumor antigen induces strong antitumor activity.

[0068]FIG. 21 is a bar graph illustrating that intravenousadministration of CLDC encoding a tumor antigen induces effectiveantitumor immunity, whereas administration of DNA encoding a tumorantigen intramuscularly or intradermally does not.

[0069]FIG. 22 is a line graph showing that intravenous administration ofCLDC encoding a tumor antigen induces a potent humoral immune responseagainst the tumor antigen in vivo.

[0070]FIG. 23 is a bar graph showing that CLDC-mediated immunizationwith a tumor antigen induces antigen-specific production of IFN* bysplenocytes.

[0071]FIG. 24 is a bar graph demonstrating that CLRC-mediatedimmunization with tumor RNA with and without DNA encoding a cytokineinduces strong antitumor activity in vivo.

[0072]FIG. 25 is a bar graph illustrating that immunization with CLRCcontaining tumor-specific RNA induces tumor-specific CTL responses invivo.

[0073]FIG. 26 is a line graph showing that intraperitoneal immunizationwith CLDC containing DNA encoding IL-2 induces a reduction in FeLV viraltiter.

[0074]FIG. 27 is a line graph illustrating that intravenous pulmonarytransfection with CLDC containing DNA encoding IFN* inhibits thedevelopment of airway hyperresponsiveness in allergen sensitized andchallenged mice.

[0075]FIG. 28 is a bar graph demonstrating that intravenous pulmonarytransfection with CLDC containing DNA encoding IFN* inhibits eosinophilinflux to the airways in mice sensitized and challenged with allergen.

[0076]FIG. 29A is a bar graph illustrating that intravenousadministration of CLDC induces IFN* release from spleen as compared tointratracheal administration.

[0077]FIG. 29B is a bar graph illustrating that intravenousadministration of CLDC induces IFN* release from lung as compared tointratracheal administration.

DETAILED DESCRIPTION OF THE INVENTION

[0078] The present invention generally relates to a novel geneticimmunization strategy and therapeutic compositions for eliciting animmune response in a mammal, and in particular, in a mammal that has adisease amenable to treatment by elicitation of an immune response.Diseases which are particularly amenable to treatment using the methodof the present invention include cancer, allergic inflammation andinfectious disease. In one embodiment, the method and composition of thepresent invention are particularly useful for the prevention andtreatment of primary lung cancers, pulmonary metastatic diseases,allergic asthma and viral diseases. In another embodiment, the methodand composition of the present invention are useful for treating chronicobstructive pulmonary diseases. In addition, elicitation of an immuneresponse according to the method of the present invention can be usefulfor the development and implementation of immunological diagnostic andresearch tools and assays.

[0079] More particularly, the genetic immunization method of the presentinvention comprises the elicitation of an immune response in a mammal byintravenous or intraperitoneal administration (i.e., systemicadministration) of a therapeutic composition that includes an isolatednucleic acid molecule complexed with a liposome delivery vehicle. Thepresent inventors have made the surprising discovery that thecombination of nucleic acids and liposomes is highly immunostimulatoryin vivo when administered by intravenous or intraperitoneal injection.The potency of this immune response is far greater than the responseinduced by administration of either nucleic acids or liposomes alone(See Examples 1b, 1h and 2b), and is dependent upon the intravenous orintraperitoneal administration of the complex (See Examples 5 and 6b).Moreover, this effect is independent of whether or not a protein isencoded by or expressed by the nucleic acids (See Examples 1 and 2), andit is also independent of the source of the nucleic acids (e.g.,mammalian, bacterial, insect, viral; see Examples 1g and 2c), the typeof nucleic acids (e.g., DNA or RNA; see Examples 7a-b), and to someextent, the type of lipids used (See Example 1f). As such, the nucleicacid-lipid complexes of the present invention induce a strong, systemic,non-antigen-specific immune response when administered intravenously orintraperitoneally, which results in the activation of multiple differentimmune effector cells in vivo. The present inventors have additionallydiscovered that the immune response generated by such a nucleicacid-lipid complex administered by the present method has potentanti-tumor, anti-allergy and anti-viral properties (See Examples 1a-c,1h-1,2a-d, 8 and 9). Immune activation induced by such a therapeuticcomposition of the present invention is quantitatively more potent thanthat induced by either LPS (endotoxin) or poly I/C (a classical inducerof antiviral immune responses; see Examples 1c and 1i). Furthermore, thetype of immune stimulation induced (e.g., as characterized by thepattern of cytokines induced) also differs qualitatively from thatinduced by LPS or poly I/C. Finally, this effect does not appear to beassociated with the complement cascade problems that have beenexperienced using viral delivery systems.

[0080] These findings are surprising because, prior to the presentinvention, liposome delivery vehicles, which are often used in genetherapy protocols, were touted by many in the art as being relativelynon-immunogenic, particularly as compared to viral vector deliveryvehicles (e.g., adenovirus vectors), and have thus been considered safeand useful for delivering a gene to a site in a mammal whilesubstantially avoiding an immune inflammatory response (See, forexample, Liu et al., 1997, Nature Biotechnology 15:167-173, Stewart etal., 1992, Hum. Gene Ther. 3:267-275; Zhu et al., 1993, Science261:209-211; Canonico et al., 1994, J. Appl. Phys. 77:415-419). Thisrecognized relative non-immunogenicity of liposomes has motivated thoseof skill in the art to use liposomes to deliver genes with theconfidence that the delivery vehicle is relatively innocuous in vivo.The present invention provides evidence that contradicts this principle.

[0081] The discovery of the present inventors is further surprisingbecause, although it was previously recognized that administration ofnaked DNA (i.e., by intramuscular or percutaneous delivery), whichcomprises a bacterially derived vector ligated to a target gene,provides an adjuvant effect (i.e., due to the bacterially derived vectorDNA), the nucleic acid:lipid complexes of the present invention aresignificantly more immunostimulatory than DNA administered alone (i.e.,naked DNA) (See Examples section). This discovery by the presentinventors is quite unexpected and thus represents a new frontier ingenetic vaccine design. Previously described naked DNA vaccines aretypically designed to use bacterial plasmid DNA, since a vast body ofliterature has reported that bacterial and some insect nucleic acids maybe immunogenic (See, for example, Pisetsky et al., 1996, Immunity,5:303-310; Pisetsky, 1996, Journal of Immunology 156:421-423; Yamamoto,et al., 1994, Microbiol. Immunol. 38(10):831-836; Roman, et al., 1997,Nature Medicine, 3(8):849-854; Krieg, 1996, Trends in Microbiology,4(2):73-77; Sun, et al., 1996, Immunity, 4:555-564; Stacey et al., 1996,The Journal of Immunology, 157:2116-2122; Sato, et al., 1996, Science,273:352-354; or Ballas, 1996, The Journal of Immunology, 157:1840-1845).Significantly, this literature has specifically excluded mammaliannucleic acids for use in naked DNA vaccines, asserting that mammaliannucleic acids are not immunogenic. Therefore, it is completelyunpredicted by the art at the time of the present invention that nucleicacids from mammalian sources would have immunostimulatory properties,and it is even more unexpected that the effect of nucleic acids from anysource complexed with lipids at very low doses would synergize toprovide such a strong immunostimulatory effect demonstrated by thepresent inventors, particularly in comparison to lipids or nucleic acidsalone.

[0082] In view of the present inventors' discoveries, previousinvestigators in the art may be misdirecting the use of liposomedelivery vehicles for gene therapy when elicitation of an immuneresponse is not desirable. Moreover, with regard to geneticimmunization, which is the primary focus of the present invention,previous investigators have not taken advantage of the superiorimmunostimulatory effect of nucleic acid:lipid complexes in designinggenetic vaccines. In fact, most of the disclosed specific geneticimmunization strategies do not make use of liposome delivery and/or areadministered by intramuscular, intradermal, oral or aerosol deliveryroutes, for the reasons discussed above.

[0083] The present inventors disclose herein that alternate,non-systemic routes of administration (i.e., other than intravenous orintraperitoneal) significantly decrease both the immunostimulatoryeffect and the therapeutic efficacy of the present composition incomparison with administration by the present method. Specifically, thepresent inventors have found that the efficacy of the geneticimmunization method of the present invention is unattainable usingpreviously described genetic immunization protocols wherein naked DNA isdelivered intramuscularly or percutaneously, even when such protocolsuse 10 to 100 times more DNA than the present method (See Example 5 and6b-c). The present inventors' discovery is surprising, because there wasno suggestion in any genetic immunization disclosure that the particulargenetic immunization protocol of the present invention would beconsiderably more efficacious than other possible protocols.

[0084] When the route of administration is intravenous, the primary siteof immunization (i.e., elicitation of an immune response) is the lung,which is a very active organ immunologically, containing large numbersof both effector cells (e.g., T cells, B cells, NK cells) and antigenpresenting cells (e.g., macrophages, dendritic cells). Similarly, whenthe route of administration is intraperitoneal, the primary sites ofimmunization are the spleen and liver, both of which are alsoimmunologically active organs. Without being bound by theory, thepresent inventors believe that these organs are capable of mounting arobust, non-antigen-specific immune response both in the tissues andsystemically, due to the mode of administration. Additionally, when thenucleic acid molecules of the nucleic acid:lipid complex encode andexpress an immunogen, these organs are further capable of expressing theimmunogen and mounting a strong antigen-specific immune response againstantigens that are encountered within the tissues. These activated immunecells are then capable of eliciting an immune response in other areas ofthe body in which the appropriate antigen is encountered. Administrationof the nucleic acid:lipid complexes can be at any site in the mammalwherein systemic administration (i.e., intravenous or intraperitonealadministration) is possible, including to sites in which the target sitefor immune activation is not the first organ having a capillary bedproximal to the site of administration.

[0085] As discussed above, the use of genetic vaccines and gene therapyvehicles has generally been described in the art (See for example, U.S.Pat. No. 5,593,972, issued Jan. 14, 1997, to Weiner et al.; U.S. Pat.No. 5,580,859, issued Dec. 3, 1996, to Felgner et al.; U.S. Pat. No.5,589,466, issued Dec. 31, 1996, to Felgner et al.; U.S. Pat. No.5,641,662, issued Jun. 24, 1997, to Debs et al. and U.S. Pat. No.5,676,954, issued Oct. 14, 1997, to Brigham). Such publications havebroadly disclosed genetic vaccine and/or gene therapy protocols whichinclude administration of nucleic acid molecules (e.g., DNA) encodingany of a variety of antigens and other proteins, which are administeredto an animal by a variety of administration routes, and using a varietyof delivery mechanisms. These disclosures have failed, however, toappreciate the surprising advantages and unexpected efficacy of theparticular genetic immunization compositions and methods discovered bythe present inventors. Indeed, in view of the above discussion, many ofthe methods and compositions for genetic immunization and/or genetherapy disclosed by the above publications are predicted to beinoperable, unsafe, and/or significantly less effective in vivo than thespecific compositions and methods of the present invention. The presentinventors' discoveries provide strong evidence that the development ofboth genetic vaccines designed to immunize an animal and gene therapyprotocols designed to deliver a gene to a site in an animal should bereevaluated to avoid previously unknown safety and efficacy concerns.

[0086] Due to the unexpected immunostimulatory properties of the nucleicacid:lipid complexes administered by the present method, the geneticimmunization method of the present invention is particularly useful inhuman treatments because traditional adjuvants can be avoided. This is aparticular advantage of the present method, since some traditionaladjuvants can be toxic (e.g., Freund's adjuvant and other bacterial cellwall components) and others are relatively ineffective (e.g.,aluminum-based salts and calcium-based salts). Moreover, the onlyadjuvants currently approved for use in humans in the United States arethe aluminum salts, aluminum hydroxide and aluminum phosphate, neitherof which stimulates cell-mediated immunity. In addition, as will beshown in the Examples below, traditional naked DNA delivery, which hasbeen touted as having an adjuvant effect, is far less effective than thepresent compositions at stimulating a non-antigen-specific immuneresponse. Finally, unlike many protocols for administration of viralvector-based genetic vaccines, the present method can be used torepeatedly deliver the therapeutic composition described herein withoutconsequences associated with some non-specific arms of the immuneresponse, such as the complement cascade.

[0087] In further embodiments of the present invention, the presentinventors have taken advantage of the non-antigen-specificimmunostimulatory effect of the above-described method and havedeveloped an even more powerful genetic immunization strategy in which anucleic acid sequence in the above nucleic acid-lipid complex encodes animmunogen and/or a cytokine that is expressed in the tissues of themammal (i.e., is operatively linked to a transcription control sequence;see Examples 4-9). The present inventors have also found that thecombination of an antigen-specific immune response elicited byexpression of an immunogen, in conjunction with the powerful,non-antigen specific immune response elicited by the nucleic acid:lipidcomplex results in a vaccine that has significantly greater in vivoefficacy than previously described genetic vaccines (See Examples 5,6b-c, 9). This effect can be additionally enhanced by coadministrationof a nucleic acid molecule encoding a cytokine such that the cytokine isexpressed in the tissues (See Examples 4 and 7a).

[0088] Moreover, with regard to intravenous administration of thepresent composition, in cancer patients, the lung is the principal siteto which metastatic tumors spread. The method of the present inventionis particularly successful in mammals having cancer, because it inducesa strong enough immune response to reduce or eliminate a primary tumorand to control any metastatic tumors that are already present, includinglarge metastatic tumors. Therefore, the genetic immunization method andcompositions of the present invention, unlike previously describedgenetic immunization methods, elicit both a systemic,non-antigen-specific immune response (similar to a conventionaladjuvant) and, when the nucleic acid encodes a tumor antigen, a strong,antigen-specific, intrapulmonary (intravenous administration; seeExamples 1e, 3 and 5) or splenic and/or hepatic (intraperitonealadministration; see Examples 1e and 1l) immune response in a mammalwhich is effective to significantly reduce or eliminate establishedtumors in vivo.

[0089] One embodiment of the present invention is a method to elicit asystemic, non-antigen-specific immune response in a mammal immuneresponse in a mammal. In this method, a therapeutic composition whichincludes: (a) a liposome delivery vehicle; and (b) an isolated nucleicacid molecule that is not operatively linked to a transcription controlsequence, is administered by intravenous or intraperitonealadministration to a mammal. Administration of such a composition by themethod of the present invention results in the elicitation of asystemic, non-antigen-specific immune response in the mammal to whichthe composition is administered. As discussed above, this immuneresponse additionally has strong, systemic, anti-tumor, anti-allergicinflammation (i.e., protective), and anti-viral properties. Suchproperties include the activation of NK cells (as measured byupregulation of NK cell markers, such as NK1.1, for example, or byproduction of IFN*), production of Th1-type cytokines (e.g., IFN*) andthe non-antigen-specific recruitment and upregulation of activity inmononuclear cells and T lymphocytes.

[0090] Therapeutic compositions useful in the method of the presentinvention include compositions containing nucleic acids having anynucleic acid sequence, including coding (i.e. encoding at least aportion of a protein or peptide) and/or non-coding (i.e., not encodingany portion of a protein or peptide) sequences, and including DNA and/orRNA. In the above-described embodiment of the present invention, sinceexpression of a protein encoded by the nucleic acid molecule is notrequired for elicitation of a systemic, non-antigen-specific immuneresponse, the molecule is not necessarily operatively linked to atranscription control sequence. It is to be noted, however, that furtheradvantages can be obtained (i.e., antigen-specific and enhancedimmunity) by including in the composition a nucleic acid sequence (DNAor RNA) which encodes an immunogen and/or a cytokine.

[0091] In another embodiment of the present invention, the presentmethod of eliciting an immune response can be modified to include theintravenous or intraperitoneal administration to a mammal of atherapeutic composition comprising: (a) a liposome delivery vehicle; and(b) a recombinant nucleic acid molecule comprising a nucleic acidsequence which encodes an immunogen. According to the present invention,the terms “immunogen” and “antigen” can be used interchangeably,although the term “antigen” is primarily used herein to describe aprotein which elicits a humoral and/or cellular immune response (i.e.,is antigenic), and the term “immunogen” is primarily used herein todescribe a protein which elicits a humoral and/or cellular immuneresponse in vivo, such that administration of the immunogen to a mammalmounts an immunogen-specific (antigen-specific) immune response againstthe same or similar proteins that are encountered within the tissues ofthe mammal. According to the present invention, an immunogen or anantigen can be any portion of a protein, naturally occurring orsynthetically derived, which elicits a humoral and/or cellular immuneresponse. As such, the size of an antigen or immunogen can be as smallas about 5-12 amino acids and as large as a full length protein,including a multimer and fusion proteins. The terms, “immunogen” and“antigen”, as used to describe the present invention, do not include asuperantigen. A superantigen is defined herein as the art-recognizedterm. More particularly, a superantigen is a molecule within a family ofproteins that binds to the extracellular portion of an MHC molecule(i.e., not in the peptide binding groove) to form and MHC:superantigencomplex. The activity of a T cell can be modified when a TCR binds to anMHC:superantigen complex. Under certain circumstances, anMHC:superantigen complex can have a mitogenic role (i.e., the ability tostimulate the proliferation of T cells) or a suppressive role (i.e.,deletion of T cell subsets).

[0092] In preferred embodiments, the immunogen is selected from thegroup of a tumor antigen, an allergen or an antigen of an infectiousdisease pathogen (i.e., a pathogen antigen). In this embodiment, thenucleic acid sequence is operatively linked to a transcription controlsequence, such that the immunogen is expressed in a tissue of a mammal,thereby eliciting an immunogen-specific immune response in the mammal,in addition to the non-specific immune response discussed above.

[0093] In a further embodiment of the method of the present invention,the therapeutic composition to be administered to a mammal includes anisolated nucleic acid molecule encoding a cytokine (also referred toherein as a “cytokine-encoding nucleic acid molecule”), in which thenucleic acid molecule is operatively linked to one or more transcriptioncontrol sequences. The result of administration of such a therapeuticcomposition to the mammal is that the nucleic acid molecule encoding thecytokine is expressed in the pulmonary tissues of the mammal, whenadministration is intravenous, and in the spleen and liver tissues ofthe mammal when administration is peritoneal. It is to be noted that theterm “a” or “an” entity refers to one or more of that entity; forexample, a cytokine refers to one or more cytokines. As such, the terms“a” (or “an”), “one or more” and “at least one” can be usedinterchangeably herein. The nucleic acid sequence encoding a cytokinecan be on the same recombinant nucleic acid molecule as a nucleic acidsequence encoding an immunogen, or on a different recombinant nucleicacid molecule.

[0094] A composition useful in the method of the present invention, asdiscussed in detail below, comprises: (a) a liposome delivery vehicle;and (b) a nucleic acid molecule, such molecule including: (1) anisolated nucleic acid sequence that is not operatively linked to atranscription control sequence; (2) an isolated non-coding nucleic acidsequence; (3) an isolated recombinant nucleic acid molecule encoding animmunogen operatively linked to a transcription control sequence,wherein the nucleic acid:lipid complex has a ratio of from about 1:1 toabout 1:64; and/or (4) an isolated recombinant nucleic acid moleculeencoding a cytokine. In preferred embodiments, the nucleic acid:lipidcomplex has a ratio of from about 1:10 to 1:40. Various components ofsuch a composition are described in detail below.

[0095] Elicitation of an immune response in a mammal can be an effectivetreatment for a wide variety of medical disorders, and in particular,for cancer, allergic inflammation and/or infectious disease. As usedherein, the term “elicit” can be used interchangeably with the terms“activate”, “stimulate”, “generate” or “upregulate”. According to thepresent invention, “eliciting an immune response” in a mammal refers tospecifically controlling or influencing the activity of the immuneresponse, and can include activating an immune response, upregulating animmune response, enhancing an immune response and/or altering an immuneresponse (such as by eliciting a type of immune response which in turnchanges the prevalent type of immune response in a mammal from one whichis harmful or ineffective to one which is beneficial or protective. Forexample, elicitation of a Th1-type response in a mammal that isundergoing a Th2-type response, or vice versa, may change the overalleffect of the immune response from harmful to beneficial. Eliciting animmune response which alters the overall immune response in a mammal canbe particularly effective in the treatment of allergic inflammation,mycobacterial infections, or parasitic infections. According to thepresent invention, a disease characterized by a Th2-type immune response(alternatively referred to as a Th2 immune response), can becharacterized as a disease which is associated with the predominantactivation of a subset of helper T lymphocytes known in the art asTh2-type T lymphocytes (or Th2 lymphocytes), as compared to theactivation of Th1-type T lymphocytes (or Th1 lymphocytes). According tothe present invention, Th2-type T lymphocytes can be characterized bytheir production of one or more cytokines, collectively known asTh2-type cytokines. As used herein, Th2-type cytokines includeinterleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6),interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-13 (IL-13) andinterleukin-15 (IL-15). In contrast, Th1-type lymphocytes producecytokines which include IL-2 and IFN*. Alternatively, a Th2-type immuneresponse can sometimes be characterized by the predominant production ofantibody isotypes which include IgG1 (the approximate human equivalentof which is IgG4) and IgE, whereas a Th1-type immune response cansometimes be characterized by the production of an IgG2a or an IgG3antibody isotype (the approximate human equivalent of which is IgG1,IgG2 or IgG3).

[0096] Preferably, the method of the present invention elicits an immuneresponse against a tumor, an allergen or an infectious disease pathogen.In particular, eliciting an immune response in a mammal refers toregulating cell-mediated immunity (i.e., helper T cell (Th) activity,cytotoxic T lymphocyte (CTL) activity, NK cell activity) and/or humoralimmunity (i.e., B cell/immunoglobulin activity), including Th1-typeand/or Th2-type cellular and/or humoral activity. In a preferredembodiment, the method of the present invention increases or elicitseffector cell immunity against a tumor, an allergen or an infectiousdisease pathogen. As used herein, effector cell immunity refers toincreasing the number and/or the activity of effector cells in themammal to which a composition is administered. In particular, T cellactivity refers to increasing the number and/or the activity of T cellsin the area of the tumor cell or pathogen. Similarly, NK cell activityrefers to increasing the number and/or activity of NK cells. In themethod of the present invention, effector cell immunity is elicited bothsystemically and in the area of the mammal in which the therapeuticcomposition is primarily targeted (i.e., intrapulmonary for intravenousadministration and in the spleen or liver for intraperitonealadministration, although the present composition is effective at othersites in the body as well). According to the present invention, aneffector cell includes a helper T cell, a cytotoxic T cell, a Blymphocyte, a macrophage, a monocyte and/or a natural killer cell. Forexample, the method of the present invention can be performed toincrease the number of effector cells in a mammal that are capable ofkilling a target cell or releasing cytokines when presented withantigens derived from a tumor cell, an allergen or a pathogen.

[0097] According to the present invention, elicitation of anon-antigen-specific immune response (i.e., a non-specific immuneresponse) includes stimulation of non-specific immune cells, such asmacrophages and neutrophils, as well as induction of cytokineproduction, particularly IFN* production, and non-antigen-specificactivation of effector cells such as NK cells, B lymphocytes and/or Tlymphocytes. More specifically, the systemic, non-antigen-specificimmune response elicited by the method and composition of the presentinvention result in an increase in natural killer (NK) cell function andnumber in the mammal, wherein an increase in NK function is defined asany detectable increase in the level of NK cell function compared to NKcell function in mammals not immunized with a composition of the presentinvention, or in mammals immunized with a composition of the presentinvention by a non-systemic (i.e., non-intravenous, non-intraperitoneal)route of administration, with the amount of nucleic acid delivered andthe ratio of nucleic acid:lipid being equal. NK function (i.e.,activity) can be measured by cytotoxicity assays against a suitabletarget cell. An example of a suitable target cell by which to measure NKcell cytotoxic activity is YAC-1. An example of an NK cell cytotoxicityassay is presented in Example 1 (FIG. 11). NK cell activation can bemeasured by determining an upregulation of NK1.1/CD69 on cells invarious organs, including spleen, lymph node, lung and liver, by flowcytometric analysis (See Example 1, FIGS. 1 and 2). Additionally, thesystemic, non-antigen-specific immune response elicited by the methodand composition of the present invention can result in an increase inproduction of IFN* by the NK cells in the mammal in various organsincluding spleen and lung, wherein an increase in IFN* production isdefined as any detectable increase in the level of IFN* productioncompared to IFN* production by NK cells in mammals not administered witha composition of the present invention, or in mammals administered witha composition of the present invention by a non-systemic route ofadministration, with the amount of nucleic acid delivered and the ratioof nucleic acid:lipid being equal. IFN* production can be measured by aIFN* ELISA (as is known in the art; Example 1, FIG. 10). Preferably, acomposition of the present invention administered by the method of thepresent invention elicits at least about 100 μg/ml of IFN* per 5×10⁶mononuclear cells from blood, spleen or lung, and more preferably, atleast about 500 μg/ml of IFN*, and more preferably at least about 1000μg/ml of IFN*, and even more preferably, at least about 5000 μg/ml ofIFN*, and even more preferably, at least about 10,000 μg/ml of IFN*.

[0098] Accordingly, the method of the present invention preferablyelicits an immune response in a mammal such that the mammal is protectedfrom a disease that is amenable to elicitation of an immune response,including cancer, allergic inflammation and/or an infectious disease. Asused herein, the phrase “protected from a disease” refers to reducingthe symptoms of the disease; reducing the occurrence of the disease,and/or reducing the severity of the disease. Protecting a mammal canrefer to the ability of a therapeutic composition of the presentinvention, when administered to a mammal, to prevent a disease fromoccurring and/or to cure or to alleviate disease symptoms, signs orcauses. As such, to protect a mammal from a disease includes bothpreventing disease occurrence (prophylactic treatment) and treating amammal that has a disease (therapeutic treatment). In particular,protecting a mammal from a disease is accomplished by eliciting animmune response in the mammal by inducing a beneficial or protectiveimmune response which may, in some instances, additionally suppress(e.g., reduce, inhibit or block) an overactive or harmful immuneresponse. The term, “disease” refers to any deviation from the normalhealth of a mammal and includes a state when disease symptoms arepresent, as well as conditions in which a deviation (e.g., infection,gene mutation, genetic defect, etc.) has occurred, but symptoms are notyet manifested.

[0099] More specifically, a therapeutic composition as described herein,when administered to a mammal by the method of the present invention,preferably produces a result which can include alleviation of thedisease, elimination of the disease, reduction of a tumor or lesionassociated with the disease, elimination of a tumor or lesion associatedwith the disease, prevention of a secondary disease resulting from theoccurrence of a primary disease (e.g., metastatic cancer resulting froma primary cancer), prevention of the disease, and stimulation ofeffector cell immunity against the disease.

[0100] One component of the therapeutic composition used in the presentmethod is a nucleic acid sequence, which can include coding and/ornon-coding nucleic acid sequences, and both oligonucleotides (describedbelow) and larger nucleic acid sequences. Although the phrase “nucleicacid molecule” primarily refers to the physical nucleic acid moleculeand the phrase “nucleic acid sequence” primarily refers to the sequenceof nucleotides on the nucleic acid molecule, the two phrases can be usedinterchangeably. As used herein, a “coding” nucleic acid sequence refersto a nucleic acid sequence which encodes at least a portion of a peptideor protein (e.g. a portion of an open reading frame), and can moreparticularly refer to a nucleic acid sequence encoding a peptide orprotein which is operatively linked to a transcription control sequence,so that the peptide or protein can be expressed. A “non-coding” nucleicacid sequence refers to a nucleic acid sequence which does not encodeany portion of a peptide or protein. According to the present invention,“non-coding” nucleic acids can include regulatory regions of atranscription unit, such as a promoter region. The term, “empty vector”can be used interchangeably with the term “non-coding,” and particularlyrefers to a nucleic acid sequence in the absence of a protein codingportion, such as a plasmid vector without a gene insert. The phrase“operatively linked” refers to linking a nucleic acid molecule to atranscription control sequence in a manner such that the molecule can beexpressed when transfected (i.e., transformed, transduced ortransfected) into a host cell. Therefore, a nucleic acid sequence thatis “not operatively linked to a transcription control sequence” refersto any nucleic acid sequence, including both coding and non-codingnucleic acid sequences, which are not linked to a transcription controlsequence in a manner such that the molecule is able to be expressed whentransfected into a host cell. It is noted that this phrase does notpreclude the presence of a transcription control sequence in the nucleicacid molecule.

[0101] In some embodiments of the present invention, a nucleic acidsequence included in a therapeutic composition of the present inventionis incorporated into a recombinant nucleic acid molecule, and encodes animmunogen and/or a cytokine. As discussed in detail below, preferredimmunogens include a tumor antigen, an allergen or an antigen from aninfectious disease pathogen (i.e., a pathogen antigen). The phrase“recombinant molecule” primarily refers to a nucleic acid molecule ornucleic acid sequence operatively linked to a transcription controlsequence, but can be used interchangeably with the phrase “nucleic acidmolecule” which is administered to a mammal.

[0102] According to the present invention, an isolated, or biologicallypure, nucleic acid molecule or nucleic acid sequence, is a nucleic acidmolecule or sequence that has been removed from its natural milieu. Assuch, “isolated” and “biologically pure” do not necessarily reflect theextent to which the nucleic acid molecule has been purified. An isolatednucleic acid molecule useful in the present composition can include DNA,RNA, or derivatives of either DNA or RNA. An isolated nucleic acidmolecule useful in the present composition can include oligonucleotidesand larger sequences, including both nucleic acid molecules that encodea protein or a fragment thereof, and nucleic acid molecules thatcomprise regulatory regions, introns, or other non-coding DNA or RNA.Typically, an oligonucleotide has a nucleic acid sequence from about 1to about 500 nucleotides, and more typically, is at least about 5nucleotides in length. Immune activation by nucleic acid:lipid complexesof the present invention can be induced by eukaryotic as well asprokaryotic nucleic acids, indicating that there is some property of thenucleic acid:lipid complexes that is inherently immune activating,regardless of the source of the nucleic acids. Therefore, the nucleicacid molecule can be derived from any source, including mammalian,bacterial, insect, or viral sources, since the present inventors havediscovered that the source of the nucleic acid does not have asignificant effect on the ability to elicit an immune response by thenucleic acid-lipid complex. In one embodiment of the present invention,the nucleic acid molecule used in a therapeutic composition of thepresent invention is not a bacterial nucleic acid molecule.

[0103] An isolated immunogen-encoding (e.g., a tumor antigen-,allergen-, or pathogen antigen-) or cytokine-encoding nucleic acidmolecule can be obtained from its natural source, either as an entire(i.e., complete) gene or a portion thereof capable of encoding: a tumorantigen protein having a B cell and/or T cell epitope, an allergenhaving a B cell and/or T cell epitope, a pathogen antigen having a Bcell and/or a T cell epitope, or a cytokine protein capable of bindingto a complementary cytokine receptor. A nucleic acid molecule can alsobe produced using recombinant DNA technology (e.g., polymerase chainreaction (PCR) amplification, cloning) or chemical synthesis. Nucleicacid molecules include natural nucleic acid molecules and homologuesthereof, including, but not limited to, natural allelic variants andmodified nucleic acid molecules in which nucleotides have been inserted,deleted, substituted, and/or inverted in such a manner that suchmodifications do not substantially interfere with the nucleic acidmolecule's ability to encode an immunogen or a cytokine useful in themethod of the present invention.

[0104] A nucleic acid molecule homologue can be produced using a numberof methods known to those skilled in the art (see, for example, Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LabsPress, 1989), which is incorporated herein by reference in its entirety.For example, nucleic acid molecules can be modified using a variety oftechniques including, but not limited to, classic mutagenesis techniquesand recombinant DNA techniques, such as site-directed mutagenesis,chemical treatment of a nucleic acid molecule to induce mutations,restriction enzyme cleavage of a nucleic acid fragment, ligation ofnucleic acid fragments, polymerase chain reaction (PCR) amplificationand/or mutagenesis of selected regions of a nucleic acid sequence,synthesis of oligonucleotide mixtures and ligation of mixture groups to“build” a mixture of nucleic acid molecules and combinations thereof.Nucleic acid molecule homologues can be selected from a mixture ofmodified nucleic acids by screening for the function of the proteinencoded by the nucleic acid (e.g., tumor antigen, allergen or pathogenantigen immunogenicity, or cytokine activity, as appropriate).Techniques to screen for immunogenicity, such as tumor antigen, allergenor pathogen antigen immunogenicity, or cytokine activity, are known tothose of skill in the art and include a variety of in vitro and in vivoassays.

[0105] As heretofore disclosed, immunogen or cytokine proteins of thepresent invention include, but are not limited to, proteins encoded bynucleic acid molecules having full-length immunogen or cytokine codingregions; proteins encoded by nucleic acid molecules having partialimmunogen regions which contain at least one T cell epitope and/or atleast one B cell epitope; proteins encoded by nucleic acid moleculeshaving cytokine coding regions capable of binding to a complementarycytokine receptor; fusion proteins; and chimeric proteins comprisingcombinations of different immunogens and/or cytokines.

[0106] One embodiment of the present invention is an isolated nucleicacid molecule that encodes at least a portion of a full-lengthimmunogen, including a tumor antigen, allergen or pathogen antigen, or ahomologue of such immunogens. As used herein, “at least a portion of animmunogen” refers to a portion of an immunogen protein containing a Tcell and/or a B cell epitope. In one embodiment, an immunogen-encodingnucleic acid molecule includes an entire coding region of such animmunogen. As used herein, a homologue of an immunogen is a proteinhaving an amino acid sequence that is sufficiently similar to a naturalimmunogen amino acid sequence (i.e., a naturally occurring, endogenous,or wild-type immunogen) that a nucleic acid sequence encoding thehomologue encodes a protein capable of eliciting an immune responseagainst the natural immunogen.

[0107] A tumor antigen-encoding nucleic acid molecule of the presentinvention encodes an antigen that can include tumor antigens havingepitopes that are recognized by T cells, tumor antigens having epitopesthat are recognized by B cells, tumor antigens that are exclusivelyexpressed by tumor cells, and tumor antigens that are expressed by tumorcells and by non-tumor cells. Preferably, tumor antigens useful in thepresent method have at least one T cell and/or B cell epitope.Therefore, expression of the tumor antigen in a tissue of a mammalelicits a tumor antigen-specific immune response against the tumor inthe tissue of the mammal. As discussed above, the present inventors havefound that administration of the nucleic acid:lipid complex of thepresent invention elicits a strong, systemic, non-antigen-specific,anti-tumor response in vivo, and this effect enhances theantigen-specific immune response to a tumor antigen expressed by thenucleic acid molecule.

[0108] In a preferred embodiment, a nucleic acid molecule of the presentinvention encodes a tumor antigen from a cancer selected from the groupof melanomas, squamous cell carcinoma, breast cancers, head and neckcarcinomas, thyroid carcinomas, soft tissue sarcomas, bone sarcomas,testicular cancers, prostatic cancers, ovarian cancers, bladder cancers,skin cancers, brain cancers, angiosarcomas, hemangiosarcomas, mast celltumors, primary hepatic cancers, lung cancers, pancreatic cancers,gastrointestinal cancers, renal cell carcinomas, hematopoieticneoplasias and metastatic cancers thereof.

[0109] According to the present invention, a pathogen antigen-encodingnucleic acid molecule of the present invention encodes an antigen froman infectious disease pathogen that can include pathogen antigens havingepitopes that are recognized by T cells, pathogen antigens havingepitopes that are recognized by B cells, pathogen antigens that areexclusively expressed by pathogens, and pathogen antigens that areexpressed by pathogens and by other cells. Preferably, pathogen antigensuseful in the present method have at least one T cell and/or B cellepitope and are exclusively expressed by pathogens (i.e., and not by theendogenous tissues of the infected mammal). Therefore, expression of thepathogen antigen in a tissue of a mammal elicits an antigen-specificimmune response against the pathogen in the tissues of the mammal aswell as systemically.

[0110] According to the present invention, a pathogen antigen includesan antigen that is expressed by a bacterium, a virus, a parasite or afungus. Preferred pathogen antigens for use in the method of the presentinvention include antigens which cause a chronic infectious disease in amammal. Particularly preferred pathogen antigens for use in the presentmethod are immunogens from immunodeficiency virus (HIV), Mycobacteriumtuberculosis, herpesvirus, papillomavirus and Candida.

[0111] In one embodiment, a pathogen antigen for use in the method orcomposition of the present invention includes an antigen from a pathogenassociated with an infectious pulmonary disease, such as tuberculosis.In a more preferred embodiment, such a pathogen antigen includes anantigen from Mycobacterium tuberculosis, and even more preferably, isMycobacterium tuberculosis antigen 85.

[0112] In another embodiment of the present invention, a pathogenantigen for use in the method or composition of the present inventionincludes an immunogen from a virus. As discussed above, the presentinventors have found that the composition and method of the presentinvention are particularly useful in the treatment of and protectionagainst viral infections. Specifically, the nucleic acid:lipid complexadministered by the method of the present invention elicits a strong,systemic, non-antigen-specific, anti-viral response in vivo, regardlessof whether or not the nucleic acid encodes or expresses an immunogen.When the nucleic acid sequence does encode a viral antigen that isoperatively linked to a transcription control sequence such that theviral antigen is expressed in a tissue of a mammal, the presentcomposition further elicits a strong, viral antigen-specific immuneresponse in addition to the above-described systemic immune response. Ina preferred embodiment, the immunogen is from a virus selected from thegroup of human immunodeficiency virus and feline immunodeficiency virus.

[0113] Another embodiment of the present invention includes anallergen-encoding nucleic acid molecule that encodes at least a portionof a full-length allergen or a homologue of the allergen protein, andincludes allergens having epitopes that are recognized by T cells,allergens having epitopes that are recognized by B cells, and allergensthat are a sensitizing agent in diseases associated with allergicinflammation. Preferred allergens to use in the therapeutic compositionof the present invention include plant pollens, drugs, foods, venoms,insect excretions, molds, animal fluids, animal hair and animal dander.

[0114] Another embodiment of the present invention includes acytokine-encoding nucleic acid molecule that encodes at least a portionof a full-length cytokine or a homologue of the cytokine protein. Asused herein, “at least a portion of a cytokine” refers to a portion of acytokine protein having cytokine activity and being capable of bindingto a cytokine receptor. Preferably, a cytokine-encoding nucleic acidmolecule includes an entire coding region of a cytokine. As used herein,a homologue of a cytokine is a protein having an amino acid sequencethat is sufficiently similar to a natural cytokine amino acid sequenceso as to have cytokine activity (i.e. activity associated with naturallyoccurring, or wild-type cytokines). In accordance with the presentinvention, a cytokine includes a protein that is capable of affectingthe biological function of another cell. A biological function affectedby a cytokine can include, but is not limited to, cell growth, celldifferentiation or cell death. Preferably, a cytokine of the presentinvention is capable of binding to a specific receptor on the surface ofa cell, thereby affecting the biological function of a cell.

[0115] A cytokine-encoding nucleic acid molecule of the presentinvention encodes a cytokine that is capable of affecting the biologicalfunction of a cell, including, but not limited to, a lymphocyte, amuscle cell, a hematopoietic precursor cell, a mast cell, a naturalkiller cell, a macrophage, a monocyte, an epithelial cell, anendothelial cell, a dendritic cell, a mesenchymal cell, a Langerhanscell, cells found in granulomas and tumor cells of any cellular origin,and more preferably a mesenchymal cell, an epithelial cell, anendothelial cell, a muscle cell, a macrophage, a monocyte, a T cell anda dendritic cell.

[0116] A preferred cytokine nucleic acid molecule of the presentinvention encodes a hematopoietic growth factor, an interleukin, aninterferon, an immunoglobulin superfamily molecule, a tumor necrosisfactor family molecule and/or a chemokine (i.e., a protein thatregulates the migration and activation of cells, particularly phagocyticcells). A more preferred cytokine nucleic acid molecule of the presentinvention encodes an interleukin. An even more preferred cytokinenucleic acid molecule useful in the method of the present inventionencodes interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12(IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), and/orinterferon-* (IFN*). A most preferred cytokine nucleic acid moleculeuseful in the method of the present invention encodes interleukin-2(IL-2), interleukin-12 (IL-12), interleukin-18 (IL-18) and/orinterferon-* (IFN*).

[0117] As will be apparent to one of skill in the art, the presentinvention is intended to apply to cytokines derived from all types ofmammals. A preferred mammal from which to derive cytokines includes amouse, a human and a domestic pet (e.g., dog, cat). A more preferredmammal from which to derive cytokines includes a dog and a human. Aneven more preferred mammal from which to derive cytokines is a human.

[0118] According to the present invention, a cytokine-encoding nucleicacid molecule of the present invention is preferably derived from thesame species of mammal as the mammal to be treated. For example, acytokine-encoding nucleic acid molecule derived from a canine (i.e.,dog) nucleic acid molecule is preferably used to treat a disease in acanine. The present invention includes a nucleic acid molecule of thepresent invention operatively linked to one or more transcriptioncontrol sequences to form a recombinant molecule. As discussed above,the phrase “operatively linked” refers to linking a nucleic acidmolecule to a transcription control sequence in a manner such that themolecule can be expressed when transfected (i.e., transformed,transduced or transfected) into a host cell. Preferably, a nucleic acidmolecule used in a composition of the present invention is operativelylinked to a transcription control sequence that allows for transientexpression of the molecule in the recipient mammal. To avoid adverseaffects of prolonged immune activation (e.g., shock, excessiveinflammation, immune tolerance), it is a preferred embodiment of thepresent invention that an immunogen or cytokine encoded by a nucleicacid molecule be expressed in the immunized mammal for about 72 hours toabout 1 month, and preferably, from about 1 week to about 1 month, andmore preferably, from about 2 weeks to about 1 month. Expression of alonger period of time than 1 month is not desired in instances whereundesirable effects associated with prolonged immune activation occur.However, if such effects do not occur for a particular composition orcan be avoided or controlled, then extended expression is acceptable. Inone embodiment, transient expression can be achieved by selection ofsuitable transcription control sequences, for example. Transcriptioncontrol sequences which are suitable for transient gene expression arediscussed below.

[0119] Transcription control sequences are sequences which control theinitiation, elongation, and termination of transcription. Particularlyimportant transcription control sequences are those which controltranscription initiation, such as promoter, enhancer, operator andrepressor sequences. Suitable transcription control sequences includeany transcription control sequence that can function in at least one ofthe recombinant cells useful in the method of the present invention. Avariety of such transcription control sequences are known to thoseskilled in the art. Preferred transcription control sequences includethose which function in mammalian, bacteria, insect cells, andpreferably in mammalian cells. More preferred transcription controlsequences include, but are not limited to, simian virus 40 (SV-40), *-actin, retroviral long terminal repeat (LTR), Rous sarcoma virus (RSV),cytomegalovirus (CMV), tac, lac, trp, trc, oxy-pro, omp/lpp, miB,bacteriophage lambda (*) (such as *PL and *PR and fusions that includesuch promoters), bacteriophage T7, T7lac, bacteriophage T3,bacteriophage SP6, bacteriophage SP01, metallothionein, alpha matingfactor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such asSindbis virus subgenomic promoters), baculovirus, Heliothis zea insectvirus, vaccinia virus and other poxviruses, herpesvirus, and adenovirustranscription control sequences, as well as other sequences capable ofcontrolling gene expression in eukaryotic cells. Additional suitabletranscription control sequences include tissue-specific promoters andenhancers (e.g., T cell-specific enhancers and promoters). Transcriptioncontrol sequences of the present invention can also include naturallyoccurring transcription control sequences naturally associated with agene encoding an immunogen, including tumor antigen, an allergen, apathogen antigen or a cytokine.

[0120] Particularly preferred transcription control sequences for use inthe present invention include promoters which allow for transientexpression of a nucleic acid molecule that is to be expressed, therebyallowing for expression of the protein encoded by the nucleic acidmolecule to be terminated after a time sufficient to elicit an immuneresponse. Adverse effects related to prolonged activation of the immunesystem can be avoided by selection of promoters and other transcriptioncontrol factors which allow for transient expression of a nucleic acidmolecule. This is yet another point of difference between the method ofthe present invention and previously described gene therapy/genereplacement protocols. Suitable promoters for use with nucleic acidmolecules encoding immunogens and/or cytokines for use in the presentinvention include cytomegalovirus (CMV) promoter and othernon-retroviral virus-based promoters such as RSV promoters, adenoviruspromoters and Simian virus promoters. LTR, tissue-specific promoters,promoters from self-replication viruses and papillomavirus promoters,which may be quite desirable in gene therapy/gene replacement protocolsbecause they provide prolonged expression of a transgene, are notpreferred transcription control sequences for use in the presentinvention.

[0121] Recombinant molecules of the present invention, which can beeither DNA or RNA, can also contain additional regulatory sequences,such as translation regulatory sequences, origins of replication, andother regulatory sequences that are compatible with the recombinantcell. In one embodiment, a recombinant molecule of the present inventionalso contains secretory signals (i.e., signal segment nucleic acidsequences) to enable an expressed immunogen or cytokine protein to besecreted from the cell that produces the protein. Suitable signalsegments include: (1) an immunogen signal segment (e.g., a tumorantigen, allergen or pathogen antigen signal segment); (2) a cytokinesignal segment; (3) or any heterologous signal segment capable ofdirecting the secretion of an immunogen and/or cytokine proteinaccording to the present invention.

[0122] Preferred recombinant molecules of the present invention includea recombinant molecule containing a nucleic acid sequence encoding animmunogen, a recombinant molecule containing a nucleic acid sequenceencoding a cytokine, or a recombinant molecule containing both a nucleicacid sequence encoding an immunogen and a nucleic acid sequence encodinga cytokine to form a chimeric recombinant molecule (i.e., the nucleicacid sequence encoding the immunogen and the nucleic acid sequenceencoding the cytokine are in the same recombinant molecule). The nucleicacid molecules contained in such recombinant chimeric molecules areoperatively linked to one or more transcription control sequences, inwhich each nucleic acid molecule contained in a chimeric recombinantmolecule can be expressed using the same or different transcriptioncontrol sequences.

[0123] One or more recombinant molecules of the present invention can beused to produce an encoded product (i.e., an immunogen protein or acytokine protein) useful in the method of the present invention. In oneembodiment, an encoded product is produced by expressing a nucleic acidmolecule as described herein under conditions effective to produce theprotein. A preferred method to produce an encoded protein is bytransfecting a host cell with one or more recombinant molecules to forma recombinant cell. Suitable host cells to transfect include anymammalian cell that can be transfected. Host cells can be eitheruntransfected cells or cells that are already transformed with at leastone nucleic acid molecule. Host cells according to the present inventioncan be any cell capable of producing an immunogen (e.g., tumor, allergenor pathogen) and/or a cytokine according to the present invention. Apreferred host cell includes a mammalian lung cells, lymphocytes, musclecells, hematopoietic precursor cells, mast cells, natural killer cells,macrophages, monocytes, epithelial cells, endothelial cells, dendriticcells, mesenchymal cells, Langerhans cells, cells found in granulomasand tumor cells of any cellular origin. An even more preferred host cellof the present invention includes mammalian mesenchymal cells,epithelial cells, endothelial cells, macrophages, monocytes, lung cells,muscle cells, T cells and dendritic cells.

[0124] According to the method of the present invention, a host cell ispreferably transfected in vivo (i.e., in a mammal) as a result ofintravenous or intraperitoneal administration to a mammal of a nucleicacid molecule complexed to a liposome delivery vehicle. Transfection ofa nucleic acid molecule into a host cell according to the presentinvention can be accomplished by any method by which a nucleic acidmolecule administered with a liposome delivery vehicle can be insertedinto the cell in vivo, and includes lipofection.

[0125] It may be appreciated by one skilled in the art that use ofrecombinant DNA technologies can improve expression of transfectednucleic acid molecules by manipulating, for example, the duration ofexpression of the transgene (i.e., recombinant nucleic acid molecule),the number of copies of the nucleic acid molecules within a host cell,the efficiency with which those nucleic acid molecules are transcribed,the efficiency with which the resultant transcripts are translated, andthe efficiency of post-translational modifications. Recombinanttechniques useful for increasing the expression of nucleic acidmolecules of the present invention include, but are not limited to,operatively linking nucleic acid molecules to high-copy number plasmids,integration of the nucleic acid molecules into one or more host cellchromosomes, addition of vector stability sequences to plasmids,increasing the duration of expression of the recombinant molecule,substitutions or modifications of transcription control signals (e.g.,promoters, operators, enhancers), substitutions or modifications oftranslational control signals (e.g., ribosome binding sites,Shine-Dalgarno sequences), modification of nucleic acid molecules of thepresent invention to correspond to the codon usage of the host cell, anddeletion of sequences that destabilize transcripts. The activity of anexpressed recombinant protein of the present invention may be improvedby fragmenting, modifying, or derivatizing nucleic acid moleculesencoding such a protein. Additionally, a nucleic acid molecule, andparticularly a plasmid portion, including transcription controlsequences, can be modified to make the nucleic acids moreimmunostimulatory, such as by the addition of CpG moieties to thenucleic acids.

[0126] One embodiment of the method of the present invention, when themammal has cancer, a therapeutic composition to be intravenouslyadministered to the mammal comprises a plurality of recombinant nucleicacid molecules, wherein each of the recombinant nucleic acid moleculescomprises a cDNA sequence, each of the cDNA sequences encoding a tumorantigen or a fragment thereof (i.e., at least a portion of a tumorantigen as defined above, preferably a portion containing a T or B cellepitope). The cDNA sequences are amplified from total RNA that has beenisolated from an autologous tumor sample. Each of the plurality of cDNAsequences is operatively linked to a transcription control sequence.Administration of such a therapeutic composition to a mammal that hascancer results in the expression of the cDNA sequences encoding thetumor antigens in the tissue of the mammal (pulmonary tissue byintravenous administration and spleen and liver by intraperitonealadministration). In a further embodiment, such a therapeutic compositioncomprises a recombinant nucleic acid molecule having a nucleic acidsequence encoding a cytokine, wherein the nucleic acid sequence isoperatively linked to a transcription control sequence. Administrationof such a therapeutic composition to a mammal results in the expressionof the nucleic acid sequence encoding the cytokine in theabove-mentioned tissues of the mammal. According to this embodiment ofthe present invention, an autologous tumor sample is derived from themammal to whom the therapeutic composition is to be administered.Therefore, the cDNA sequences in the therapeutic composition will encodetumor antigens present in the cancer against which an immune response isto be elicited. In this embodiment, it is not necessary to know which ofthe antigens in a given tumor sample is the most immunogenic (i.e., thebest immunogens), since substantially all of the antigens expressed bythe tumor sample are administered to the mammal. In addition, elicitingan immune response against multiple tumor antigens/immunogens is likelyto have the benefit of enhancing the therapeutic efficacy of the immuneresponse against the cancer.

[0127] In this embodiment of the method of the present invention, aplurality of recombinant nucleic acid molecules as described can also bereferred to as a library of nucleic acid molecules, and moreparticularly, a cDNA library. Methods to produce cDNA libraries are wellknown in the art. Such methods are disclosed, for example, in Sambrooket al., supra. More particularly, in this embodiment, a therapeuticcomposition includes a plurality of recombinant cDNA molecules encodingtumor antigens, or fractions thereof, which represents the genes thatare expressed by an autologous tumor sample. Such a plurality ofrecombinant nucleic acid molecules can be produced, for example byisolating total RNA from an autologous tumor sample, converting (i.e.,amplifying) the RNA into a plurality of cDNA molecules, and thenpreparing a cDNA library by cloning the cDNA molecules into recombinantvectors to form a plurality of recombinant molecules. As used herein,total RNA refers to all of the RNA isolatable from a cellular sampleusing standard methods known in the art, and typically includes mRNA,hnRNA, tRNA and rRNA. Methods for isolating total RNA from a cellularsample, such as a tumor sample, are known in the art (See for example,Sambrook et al., supra). In a further embodiment, prior to amplificationof cDNA from the total RNA, the RNA can be selected to isolate poly-ARNA (i.e., RNA comprising a poly-A tail at the 3′ terminus, reflectiveof mRNA, the primary RNA transcript which encodes a protein expressed bya cell). In yet another embodiment, such a cDNA library can be“subtracted” against a cDNA library from a normal cellular sample in themammal in order to remove nucleic acid molecules encoding antigenspresent in non-tumor cells (i.e., normal cells) of the mammal, therebyenriching the tumor-specific immune response and preventing deleteriousimmune responses. Methods for subtraction of a nucleic acid library arealso known in the art (See Sambrook et al., supra).

[0128] In yet another embodiment of the present invention of the methodto elicit an immune response in a mammal that has cancer, a therapeuticcomposition to be intravenously or intraperitoneally administered to amammal comprises a plurality of recombinant nucleic acid molecules,wherein each of the recombinant nucleic acid molecules comprises a cDNAsequence, each of the cDNA sequences encoding a tumor antigen or afragment thereof (i.e., at least a portion of a tumor antigen as definedabove). In this embodiment, the cDNA sequences are amplified from totalRNA that has been isolated from a plurality of allogeneic tumor samplesof the same histological tumor type. Each of the plurality of cDNAsequences is operatively linked to a transcription control sequence.Administration of such a therapeutic composition to a mammal that hascancer results in the expression of the cDNA sequences encoding thetumor antigens in the tissue of the mammal (according to the route ofadministration, as previously discussed). In a further embodiment, sucha therapeutic composition comprises a recombinant nucleic acid moleculehaving a nucleic acid sequence encoding a cytokine, wherein the nucleicacid sequence is operatively linked to a transcription control sequence.Administration of such a therapeutic composition to a mammal results inthe expression of the nucleic acid sequence encoding the cytokine in thetissues of the mammal.

[0129] In this embodiment of the present invention, a plurality ofrecombinant nucleic acid molecules comprising cDNA sequences encodingtumor antigens (i.e., a cDNA library) is prepared from the total RNAisolated from a plurality of allogeneic tumor samples of the samehistological tumor type. According to the present invention, a pluralityof allogeneic tumor samples are tumor samples of the same histologicaltumor type, isolated from two or more mammals of the same species whodiffer genetically at least within the major histocompatibility complex(MHC), and typically at other genetic loci. Therefore, the plurality ofrecombinant molecules encoding tumor antigens is representative of thesubstantially all of the tumor antigens present in any of theindividuals from which the RNA was isolated. This embodiment of themethod of the present invention provides a genetic vaccine whichcompensates for natural variations between individual patients in theexpression of tumor antigens from tumors of the same histological tumortype. Therefore, administration of this therapeutic composition iseffective to elicit an immune response against a variety of tumorantigens such that the same therapeutic composition can be administeredto a variety of different individuals. Such a therapeutic compositiondelivered by the present method is particularly useful as a treatment,but may also be useful as a preventative (i.e., prophylactic) therapy.Methods to prepare such a cDNA library from a plurality of allogeneictumor samples are the same as those described above for autologous tumorsamples.

[0130] In yet another embodiment of the present invention of the methodto elicit an immune response in a mammal, a therapeutic composition tobe intravenously or intraperitoneally administered to a mammal comprisesa plurality of recombinant nucleic acid molecules, wherein each of therecombinant nucleic acid molecules comprises a cDNA sequence, each ofthe cDNA sequences encoding an immunogen from an infectious diseasepathogen or a fragment thereof (i.e., at least a portion of a pathogenantigen as defined above). In this embodiment, the cDNA sequences areamplified from total RNA that has been isolated from an infectiousdisease pathogen. Each of the plurality of cDNA sequences is operativelylinked to a transcription control sequence. Administration of such atherapeutic composition to a mammal that has or might contract aninfectious disease results in the expression of the cDNA sequencesencoding the pathogen antigens in the tissue of the mammal (according tothe route of administration, as previously discussed). In a furtherembodiment, such a therapeutic composition comprises a recombinantnucleic acid molecule having a nucleic acid sequence encoding acytokine, wherein the nucleic acid sequence is operatively linked to atranscription control sequence. Administration of such a therapeuticcomposition to a mammal results in the expression of the nucleic acidsequence encoding the cytokine in the tissues of the mammal.

[0131] In this embodiment of the present invention, the plurality ofrecombinant molecules encoding pathogen antigens is representative ofthe substantially all of the antigens present in the infectious diseasepathogen from which the RNA was isolated. In this embodiment, it is notnecessary to know which of the antigens in a given pathogen is the mostimmunogenic (i.e., the best immunogens), since substantially all of theantigens expressed by the pathogen are administered to the mammal. Inaddition, eliciting an immune response against multiple pathogenantigens/immunogens is likely to have the benefit of enhancing thetherapeutic efficacy of the immune response against the infectiousdisease. Methods to prepare such a cDNA library from an infectiousdisease pathogen are the same as those described above for tumorsamples.

[0132] In yet another embodiment of the present invention of the methodto elicit an immune response in a mammal, a therapeutic composition tobe intravenously or intraperitoneally administered to a mammal comprisesa plurality of recombinant nucleic acid molecules, each of therecombinant nucleic acid molecules comprising a cDNA sequence amplifiedfrom total RNA isolated from at least one allergen. In this embodiment,the cDNA sequences are amplified from total RNA, or a fragment thereof,that has been isolated from at least one, and preferably, multiple,allergens. Each of the plurality of cDNA sequences is operatively linkedto a transcription control sequence. Administration of such atherapeutic composition to a mammal that has or might contract a diseaseassociated with allergic inflammation results in the expression of thecDNA sequences encoding the allergens in the tissue of the mammal(according to the route of administration, as previously discussed). Ina further embodiment, such a therapeutic composition comprises arecombinant nucleic acid molecule having a nucleic acid sequenceencoding a cytokine, wherein the nucleic acid sequence is operativelylinked to a transcription control sequence. Administration of such atherapeutic composition to a mammal results in the expression of thenucleic acid sequence encoding the cytokine in the tissues of themammal. In this embodiment of the present invention, the plurality ofrecombinant molecules encoding allergens is representative of thesubstantially all of the epitopes present in the allergen from which theRNA was isolated. Additionally, more than one allergen can beadministered simultaneously.

[0133] Another embodiment of the present invention relates to a methodto elicit a tumor antigen-specific immune response and a systemic,non-specific immune response in a mammal that has cancer, which includesthe step of intravenously or intraperitoneally administering to themammal a therapeutic composition which includes: (a) a liposome deliveryvehicle; and (b) total RNA isolated from a tumor sample, wherein the RNAencodes tumor antigens or fragments thereof. Administration of such atherapeutic composition to the mammal results in the expression of theRNA encoding tumor antigens or fragments thereof in the tissue of themammal. In a preferred embodiment, the RNA is enriched for poly-A RNAprior to administration of the therapeutic composition to the mammal, asdescribed above. In a further embodiment, the therapeutic compositioncomprises a recombinant nucleic acid molecule having a nucleic acidsequence encoding a cytokine, wherein the nucleic acid sequence isoperatively linked to a transcription control sequence. Administrationof such a therapeutic composition to a mammal results in expression ofthe nucleic acid sequence encoding the cytokine in the tissue of themammal.

[0134] In this embodiment of the present invention, total RNA or morepreferably, poly-A enriched RNA, is isolated from a tumor sample aspreviously described (See Sambrook et al., supra), complexed with aliposome delivery vehicle and administered intravenously orintraperitoneally to a mammal that has cancer. The RNA encodingsubstantially all of the tumor antigens of the tumor sample is thenexpressed in the tissues of the mammal. Although RNA is normallydegraded rapidly in serum by RNAses, the present inventors believe thatRNA complexed to cationic lipids are protected from such RNAses until itreaches the tissues, where gene expression occurs. The advantage ofadministering RNA directly to a mammal according to this particularembodiment of the method of the present invention is that an immuneresponse can be elicited against multiple tumor antigens directly invivo, without requiring any substantial in vitro manipulations of thetumor tissues or host immune cells. Specific examples of this embodimentof the present invention are described in Examples 7a and 7b.

[0135] Another embodiment of the present invention relates to a methodto elicit a pathogen antigen-specific immune response and a systemic,non-specific immune response in a mammal that has an infectious disease,which includes the step of intravenously or intraperitoneallyadministering to the mammal a therapeutic composition which includes:(a) a liposome delivery vehicle; and (b) total RNA isolated from aninfectious disease pathogen, wherein the RNA encodes pathogen antigensor fragments thereof. Administration of such a therapeutic compositionto the mammal results in the expression of the RNA encoding pathogenantigens or fragments thereof in the tissue of the mammal. In apreferred embodiment, the RNA is enriched for poly-A RNA prior toadministration of the therapeutic composition to the mammal, asdescribed above. In a further embodiment, the therapeutic compositioncomprises a recombinant nucleic acid molecule having a nucleic acidsequence encoding a cytokine, wherein the nucleic acid sequence isoperatively linked to a transcription control sequence. Administrationof such a therapeutic composition to a mammal results in expression ofthe nucleic acid sequence encoding the cytokine in the tissue of themammal.

[0136] Another embodiment of the present invention relates to a methodto elicit an allergen-specific immune response and a systemic,non-specific immune response in a mammal that has a disease associatedwith allergic inflammation, which includes the step of intravenously orintraperitoneally administering to the mammal a therapeutic compositionwhich includes: (a) a liposome delivery vehicle; and (b) total RNAisolated from an allergen, wherein the RNA encodes at least one allergenprotein or a fragment thereof. Administration of such a therapeuticcomposition to the mammal results in the expression of the RNA encodingat least one allergen or a fragment thereof in the tissue of the mammal.In a preferred embodiment, the RNA is enriched for poly-A RNA prior toadministration of the therapeutic composition to the mammal, asdescribed above. In a further embodiment, the therapeutic compositioncomprises a recombinant nucleic acid molecule having a nucleic acidsequence encoding a cytokine, wherein the nucleic acid sequence isoperatively linked to a transcription control sequence. Administrationof such a therapeutic composition to a mammal results in expression ofthe nucleic acid sequence encoding the cytokine in the tissue of themammal.

[0137] A therapeutic composition of the present invention includes aliposome delivery vehicle. According to the present invention, aliposome delivery vehicle comprises a lipid composition that is capableof preferentially delivering a therapeutic composition of the presentinvention to the pulmonary tissues in a mammal when administration isintravenous, and to the spleen and liver tissues of a mammal whenadministration is intraperitoneal. The phrase “preferentiallydelivering” means that although the liposome can deliver a nucleic acidmolecule to sites other than the pulmonary or spleen and liver tissue ofthe mammal, these tissues are the primary site of delivery.

[0138] A liposome delivery vehicle of the present invention can bemodified to target a particular site in a mammal, thereby targeting andmaking use of a nucleic acid molecule of the present invention at thatsite. Suitable modifications include manipulating the chemical formulaof the lipid portion of the delivery vehicle. Manipulating the chemicalformula of the lipid portion of the delivery vehicle can elicit theextracellular or intracellular targeting of the delivery vehicle. Forexample, a chemical can be added to the lipid formula of a liposome thatalters the charge of the lipid bilayer of the liposome so that theliposome fuses with particular cells having particular chargecharacteristics. Other targeting mechanisms, such as targeting byaddition of exogenous targeting molecules to a liposome (i.e.,antibodies) are not a necessary component of the liposome deliveryvehicle of the present invention, since effective immune activation atimmunologically active organs is already provided by the composition androute of delivery of the present compositions without the aid ofadditional targeting mechanisms. Additionally, for efficacy, the presentinvention does not require that a protein encoded by a given nucleicacid molecule be expressed within the target cell (e.g., tumor cell,pathogen, etc.). The compositions and method of the present inventionare efficacious when the proteins are expressed in the vicinity of(i.e., adjacent to) the target site, including when the proteins areexpressed by non-target cells.

[0139] A liposome delivery vehicle is preferably capable of remainingstable in a mammal for a sufficient amount of time to deliver a nucleicacid molecule of the present invention to a preferred site in themammal. A liposome delivery vehicle of the present invention ispreferably stable in the mammal into which it has been administered forat least about 30 minutes, more preferably for at least about 1 hour andeven more preferably for at least about 24 hours.

[0140] A liposome delivery vehicle of the present invention comprises alipid composition that is capable of fusing with the plasma membrane ofthe targeted cell to deliver a nucleic acid molecule into a cell.Preferably, when a nucleic acid:liposome complex of the presentinvention is administered intravenously, the transfection efficiency ofa nucleic acid:liposome complex of the present invention is at leastabout 1 picogram (pg) of protein expressed per milligram (mg) of totaltissue protein per microgram (μg) of nucleic acid delivered. Morepreferably, the transfection efficiency of a nucleic acid:liposomecomplex of the present invention is at least about 10 pg of proteinexpressed per mg of total tissue protein per μg of nucleic aciddelivered; and even more preferably, at least about 50 pg of proteinexpressed per mg of total tissue protein per μg of nucleic aciddelivered; and most preferably, at least about 100 pg of proteinexpressed per mg of total tissue protein per μg of nucleic aciddelivered. When the route of delivery of a nucleic acid:lipid complex ofthe present invention is intraperitoneal, the transfection efficiency ofthe complex can be as low as 1 fg of protein expressed per mg of totaltissue protein per μg of nucleic acid delivered, with the above amountsbeing more preferred.

[0141] A preferred liposome delivery vehicle of the present invention isbetween about 100 and 500 nanometers (nm), more preferably between about150 and 450 nm and even more preferably between about 200 and 400 nm indiameter.

[0142] Suitable liposomes for use with the present invention include anyliposome. Preferred liposomes of the present invention include thoseliposomes commonly used in, for example, gene delivery methods known tothose of skill in the art. Preferred liposome delivery vehicles comprisemultilamellar vesicle (MLV) lipids and extruded lipids. Methods forpreparation of MLV's are well known in the art and are described, forexample, in the Examples section. According to the present invention,“extruded lipids” are lipids which are prepared similarly to MLV lipids,but which are subsequently extruded through filters of decreasing size,as described in Templeton et al., 1997, Nature Biotech., 15:647-652,which is incorporated herein by reference in its entirety. Althoughsmall unilamellar vesicle (SUV) lipids can be used in the compositionand method of the present invention, the present inventors have foundthat multilamellar vesicle lipids are significantly moreimmunostimulatory than SUVs when complexed with nucleic acids in vivo(See Example 2d). More preferred liposome delivery vehicles compriseliposomes having a polycationic lipid composition (i.e., cationicliposomes) and/or liposomes having a cholesterol backbone conjugated topolyethylene glycol. Preferred cationic liposome compositions include,but are not limited to DOTMA and cholesterol, DOTAP and cholesterol,DOTIM and cholesterol, and DDAB and cholesterol. A most preferredliposome composition for use as a delivery vehicle in the method of thepresent invention includes DOTAP and cholesterol.

[0143] Complexing a liposome with a nucleic acid molecule of the presentinvention can be achieved using methods standard in the art (see, forexample, methods Section A described in the Examples). According to thepresent invention a cationic lipid:DNA complex is also referred toherein as a CLDC, and a cationic lipid:RNA complex is also referred toherein as CLRC. A suitable concentration of a nucleic acid molecule ofthe present invention to add to a liposome includes a concentrationeffective for delivering a sufficient amount of nucleic acid moleculeinto a mammal such that a systemic immune response is elicited. When thenucleic acid molecule encodes an immunogen or a cytokine, a suitableconcentration of nucleic acid molecule to add to a liposome includes aconcentration effective for delivering a sufficient amount of nucleicacid molecule into a cell such that the cell can produce sufficientimmunogen and/or cytokine protein to regulate effector cell immunity ina desired manner. Preferably, from about 0.1 μg to about 10 μg ofnucleic acid molecule of the present invention is combined with about 8nmol liposomes, more preferably from about 0.5 μg to about 5 μg ofnucleic acid molecule is combined with about 8 nmol liposomes, and evenmore preferably about 1.0 μg of nucleic acid molecule is combined withabout 8 nmol liposomes. In one embodiment, the ratio of nucleic acids tolipids (μg nucleic acid:nmol lipids) in a composition of the presentinvention is preferably at least about 1:1 nucleic acid:lipid by weight(i.e., 1 μg nucleic acid:1 mmol lipid), and more preferably, at leastabout 1:5, and more preferably at least about 1:10, and even morepreferably at least about 1:20. Ratios expressed herein are based on theamount of cationic lipid in the composition, and not on the total amountof lipid in the composition. In another embodiment, the ratio of nucleicacids to lipids in a composition of the present invention is preferablyfrom about 1:1 to about 1:64 nucleic acid:lipid by weight; and morepreferably, from about 1:5 to about 1:50 nucleic acid:lipid by weight;and even more preferably, from about 1:10 to about 1:40 nucleicacid:lipid by weight; and even more preferably, from about 1:15 to about1:30 nucleic acid:lipid by weight. Another particularly preferred ratioof nucleic acid:lipid is from about 1:8 to 1:16, with 1:8 to 1:32 beingmore preferred. Typically, while non-systemic routes of nucleic acidadministration (i.e., intramuscular, intratracheal, intradermal) woulduse a ratio of about 1:1 to about 1:3, systemic routes of administrationaccording to the present invention can use much less nucleic acid ascompared to lipid and achieve equivalent or better results thannon-systemic routes. Moreover, compositions designed for genetherapy/gene replacement, even when administered by intravenousadministration, typically use more nucleic acid (e.g., from 6:1 to 1:10,with 1:10 being the least amount of DNA used) as compared to thesystemic immune activation composition and method of the presentinvention.

[0144] In another embodiment of the present invention, a therapeuticcomposition further comprises a pharmaceutically acceptable excipient.As used herein, a pharmaceutically acceptable excipient refers to anysubstance suitable for delivering a therapeutic composition useful inthe method of the present invention to a suitable in vivo site.Preferred pharmaceutically acceptable excipients are capable ofmaintaining a nucleic acid molecule of the present invention in a formthat, upon arrival of the nucleic acid molecule to a cell, the nucleicacid molecule is capable of entering the cell and being expressed by thecell if the nucleic acid molecule encodes a protein to be expressed.Suitable excipients of the present invention include excipients orformularies that transport, but do not specifically target a nucleicacid molecule to a cell (also referred to herein as non-targetingcarriers). Examples of pharmaceutically acceptable excipients include,but are not limited to water, phosphate buffered saline, Ringer'ssolution, dextrose solution, serum-containing solutions, Hank'ssolution, other aqueous physiologically balanced solutions, oils, estersand glycols. Aqueous carriers can contain suitable auxiliary substancesrequired to approximate the physiological conditions of the recipient,for example, by enhancing chemical stability and isotonicity.Particularly preferred excipients include non-ionic diluents, with apreferred non-ionic buffer being 5% dextrose in water (DW5).

[0145] Suitable auxiliary substances include, for example, sodiumacetate, sodium chloride, sodium lactate, potassium chloride, calciumchloride, and other substances used to produce phosphate buffer, Trisbuffer, and bicarbonate buffer. Auxiliary substances can also includepreservatives, such as thimerosal, m- or o-cresol, formalin and benzolalcohol. Therapeutic compositions of the present invention can besterilized by conventional methods and/or lyophilized.

[0146] According to the present invention, an effective administrationprotocol (i.e., administering a therapeutic composition in an effectivemanner) comprises suitable dose parameters and modes of administrationthat result in elicitation of an immune response in a mammal that has adisease, preferably so that the mammal is protected from the disease.Effective dose parameters can be determined using methods standard inthe art for a particular disease. Such methods include, for example,determination of survival rates, side effects (i.e., toxicity) andprogression or regression of disease. In particular, the effectivenessof dose parameters of a therapeutic composition of the present inventionwhen treating cancer can be determined by assessing response rates. Suchresponse rates refer to the percentage of treated patients in apopulation of patients that respond with either partial or completeremission. Remission can be determined by, for example, measuring tumorsize or microscopic examination for the presence of cancer cells in atissue sample.

[0147] In accordance with the present invention, a suitable single dosesize is a dose that is capable of eliciting an immune response in amammal with a disease when administered one or more times over asuitable time period. Doses can vary depending upon the disease beingtreated. In the treatment of cancer, a suitable single dose can bedependent upon whether the cancer being treated is a primary tumor or ametastatic form of cancer. Doses of a therapeutic composition of thepresent invention suitable for use with intravenous or intraperitonealadministration techniques can be used by one of skill in the art todetermine appropriate single dose sizes for systemic administrationbased on the size of a mammal.

[0148] In a preferred embodiment, an appropriate single dose of anucleic acid:liposome complex of the present invention is from about 0.1μg to about 100 μg per kg body weight of the mammal to which the complexis being administered. In another embodiment, an appropriate single doseis from about 1 μg to about 10 μg per kg body weight. In anotherembodiment, an appropriate single dose of nucleic acid:lipid complex isat least about 0.1 μg of nucleic acid to the mammal, more preferably atleast about 11g of nucleic acid, even more preferably at least about 10μg of nucleic acid, even more preferably at least about 50 μg of nucleicacid, and even more preferably at least about 100 μg of nucleic acid tothe mammal.

[0149] Preferably, when nucleic acid:liposome complex of the presentinvention contains a nucleic acid molecule which is to be expressed inthe mammal, an appropriate single dose of a nucleic acid:liposomecomplex of the present invention results in at least about 1 pg ofprotein expressed per mg of total tissue protein per μg of nucleic aciddelivered. More preferably, an appropriate single dose of a nucleicacid:liposome complex of the present invention is a dose which resultsin at least about 10 pg of protein expressed per mg of total tissueprotein per μg of nucleic acid delivered; and even more preferably, atleast about 50 pg of protein expressed per mg of total tissue proteinper μg of nucleic acid delivered; and most preferably, at least about100 pg of protein expressed per mg of total tissue protein per μg ofnucleic acid delivered. When the route of delivery of a nucleicacid:lipid complex of the present invention is intraperitoneal, anappropriate single dose of a nucleic acid:liposome complex of thepresent invention is a dose which results in as low as 1 fg of proteinexpressed per mg of total tissue protein per μg of nucleic aciddelivered, with the above amounts being more preferred.

[0150] A suitable single dose of a therapeutic composition of thepresent invention to elicit a systemic, non-antigen-specific immuneresponse in a mammal is a sufficient amount of a nucleic acid moleculecomplexed to a liposome delivery vehicle, when administeredintravenously or intraperitoneally, to elicit a cellular and/or humoralimmune response in vivo in a mammal, as compared to a mammal which hasnot been administered with the therapeutic composition of the presentinvention (i.e., a control mammal). Preferred dosages of nucleic acidmolecules to be included in a nucleic acid:lipid complex of the presentinvention have been discussed above.

[0151] A suitable single dose of a therapeutic composition to elicit animmune response against a tumor is a sufficient amount of a tumorantigen-encoding recombinant molecule, alone or in combination with acytokine-encoding recombinant molecule, to reduce, and preferablyeliminate, the tumor following lipofection of the recombinant moleculesinto cells of the tissue of the mammal that has cancer.

[0152] According to the present invention, a single dose of atherapeutic composition useful to elicit an immune response against aninfectious disease and/or against a lesion associated with such adisease, comprising a pathogen-encoding recombinant molecule combinedwith liposomes, alone or in combination with a cytokine-encodingrecombinant molecule with liposomes, is substantially similar to thosedoses used to treat a tumor (as described in detail above). Similarly, asingle dose of a therapeutic composition useful to elicit an immuneresponse against an allergen, comprising an allergen-encodingrecombinant molecule combined with liposomes, alone or in combinationwith a cytokine-encoding recombinant molecule with liposomes, issubstantially similar to those doses used to treat a tumor.

[0153] It will be obvious to one of skill in the art that the number ofdoses administered to a mammal is dependent upon the extent of thedisease and the response of an individual patient to the treatment. Forexample, a large tumor may require more doses than a smaller tumor. Insome cases, however, a patient having a large tumor may require fewerdoses than a patient with a smaller tumor, if the patient with the largetumor responds more favorably to the therapeutic composition than thepatient with the smaller tumor. Thus, it is within the scope of thepresent invention that a suitable number of doses includes any numberrequired to treat a given disease.

[0154] It is to be noted that the method of the present inventionfurther differs from previously described gene therapy/gene replacementprotocols, because the time between administration and boosting of thenucleic acid:lipid complex is significantly longer than the typicaladministration protocol for gene therapy/gene replacement. For example,elicitation of an immune response using the compositions and methods ofthe present invention typically includes an initial administration ofthe therapeutic composition, followed by booster immunizations at 3-4weeks after the initial administration, optionally followed bysubsequent booster immunizations every 3-4 weeks after the firstbooster, as needed to treat a disease according to the presentinvention. In contrast, gene therapy/gene replacement protocolstypically require more frequent administration of a nucleic acid inorder to obtain sufficient gene expression to generate or replace thedesired gene function (e.g., weekly administrations).

[0155] A preferred number of doses of a therapeutic compositioncomprising a tumor antigen-encoding recombinant molecule, alone or incombination with a cytokine-encoding recombinant molecule, complexedwith a liposome delivery vehicle in order to elicit an immune responseagainst a metastatic cancer, is from about 2 to about 10 administrationspatient, more preferably from about 3 to about 8 administrations perpatient, and even more preferably from about 3 to about 7administrations per patient. Preferably, such administrations are givenonce every 3-4 weeks, as described above, until signs of remissionappear, and then once a month until the disease is gone.

[0156] According to the present invention, the number of doses of atherapeutic composition to elicit an immune response against aninfectious disease and/or a lesion associated with such disease,comprising a pathogen antigen-encoding recombinant molecule, alone or incombination with a cytokine-encoding recombinant molecule, complexedwith a liposome delivery vehicle, is substantially similar to thosenumber of doses used to treat a tumor (as described in detail above).

[0157] A therapeutic composition is administered to a mammal in afashion to elicit a systemic, non-antigen-specific immune response in amammal, and when the nucleic acid molecule in the composition encodes animmunogen, to enable expression of the administered recombinant moleculeof the present invention into an immunogenic protein (in the case of thetumor, pathogen antigen or allergen) or immunoregulatory protein (in thecase of the cytokine) in the mammal to be treated for disease. Accordingto the method of the present invention, a therapeutic composition isadministered by intravenous or intraperitoneal injection, andpreferably, intravenously. Intravenous injections can be performed usingmethods standard in the art. According to the method of the presentinvention, administration of the nucleic acid:lipid complexes can be atany site in the mammal wherein systemic administration (i.e.,intravenous or intraperitoneal administration) is possible, particularlywhen the liposome delivery vehicle comprises cationic liposomes.Administration at any site in a mammal will elicit a potent immuneresponse when either intravenous or intraperitoneal administration isused, and particularly, when intravenous administration is used.Suitable sites for administration include sites in which the target sitefor immune activation is not restricted to the first organ having acapillary bed proximal to the site of administration (i.e., compositionscan be administered at an administration site that is distal to thetarget immunization site). In other words, for example, intravenousadministration of a composition of the present invention which is usedto treat a kidney tumor in a mammal can be administered intravenously atany site in the mammal and will still elicit a strong anti-tumor immuneresponse and be efficacious at reducing or eliminating the tumor, eventhough the kidney is not the first organ having a capillary bed proximalto the site of administration. When a specific anti-tumor effect isdesired (i.e., reduction or elimination of a tumor) and the route ofadministration is intravenous, the site of administration again can beat any site by which a composition can be administered intravenously,regardless of the location of the tumor relative to the site ofadministration. For intraperitoneal administration with regard toanti-tumor efficacy (but not immune activation/immunization), it ispreferable to use this mode of administration when the tumor is in theperitoneal cavity, or when the tumor is a small tumor. For immunizationand immune activation, as discussed above, intraperitonealadministration is a suitable mode of administration, particularly incomparison to non-systemic routes, as demonstrated in the Examplessection.

[0158] In the method of the present invention, therapeutic compositionscan be administered to any member of the Vertebrate class, Mammalia,including, without limitation, primates, rodents, livestock and domesticpets. Livestock include mammals to be consumed or that produce usefulproducts (e.g., sheep for wool production). Preferred mammals to protectinclude humans, dogs, cats, mice, rats, sheep, cattle, horses and pigs,with humans and dogs being particularly preferred, and humans being mostpreferred. While a therapeutic composition of the present invention iseffective to elicit an immune response against a disease in inbredspecies of mammals, the composition is particularly useful for elicitingan immune response in outbred species of mammals.

[0159] As discussed above, a therapeutic composition of the presentinvention administered by the present method is useful for eliciting animmune response in a mammal having a variety of diseases, andparticularly cancer, allergic inflammation and infectious diseases. Atherapeutic composition of the present invention, when deliveredintravenously or intraperitoneally, is advantageous for eliciting animmune response in a mammal that has cancer in that the compositionovercomes the mechanisms by which cancer cells avoid immune elimination(i.e., by which cancer cells avoid the immune response effected by themammal in response to the disease). Cancer cells can avoid immuneelimination by, for example, being only slightly immunogenic, modulatingcell surface antigens and inducing immune suppression. A suitabletherapeutic composition for use in eliciting an immune response in amammal that has cancer comprises a nucleic acid:lipid complex of thepresent invention, wherein the nucleic acid either is not operativelylinked to a transcription control sequence, or more preferably, encodesa tumor antigen-encoding recombinant molecule operatively linked to atranscription control sequence, alone or in combination with acytokine-encoding recombinant molecule (separately or together). Atherapeutic composition of the present invention, elicits a systemic,non-specific immune response in the mammal and, upon entering targetedpulmonary or spleen and liver cells, leads to the production of tumorantigen (and, in particular embodiments, cytokine protein) that activatecytotoxic T cells, natural killer cells, T helper cells and macrophages.Such cellular activation overcomes the otherwise relative lack of immuneresponse to cancer cells, leading to the destruction of such cells.

[0160] A therapeutic composition of the present invention which includesa nucleic acid molecule encoding a tumor antigen is useful for elicitingan immune response in a mammal that has cancer, including both tumorsand metastatic forms of cancer. Treatment with the therapeuticcomposition overcomes the disadvantages of traditional treatments formetastatic cancers. For example, compositions of the present inventioncan target dispersed metastatic cancer cells that cannot be treatedusing surgical methods. In addition, administration of such compositionsdo not result in the harmful side effects caused by chemotherapy andradiation therapy, and can be administered repeatedly. Moreover, thecompositions administered by the method of the present inventiontypically target the vesicles of tumors, so that expression of a tumorantigen or cytokine within the tumor cell itself is not necessary toprovide efficacy against the tumor. Indeed, a general advantage of thepresent invention is that delivery of the composition itself elicits apowerful immune response and expression of the nucleic acid molecule atleast in the vicinity of the target site (at or adjacent to the site)provides effective immune activation and efficacy against the target.

[0161] A therapeutic composition of the present invention which includesa nucleic acid molecule encoding a tumor antigen is preferably used toelicit an immune response in a mammal that has a cancer which includes,but is not limited to, melanomas, squamous cell carcinoma, breastcancers, head and neck carcinomas, thyroid carcinomas, soft tissuesarcomas, bone sarcomas, testicular cancers, prostatic cancers, ovariancancers, bladder cancers, skin cancers, brain cancers, angiosarcomas,hemangiosarcomas, mast cell tumors, primary hepatic cancers, lungcancers, pancreatic cancers, gastrointestinal cancers, renal cellcarcinomas, hematopoietic neoplasias, and metastatic cancers thereof.Particularly preferred cancers to treat with a therapeutic compositionof the present invention include primary lung cancers and pulmonarymetastatic cancers. A therapeutic composition of the present inventionis useful for eliciting an immune response in a mammal to treat tumorsthat can form in such cancers, including malignant and benign tumors.Preferably, expression of the tumor antigen in a pulmonary tissue of amammal that has cancer (i.e., by intravenous delivery) produces a resultselected from the group of alleviation of the cancer, reduction of atumor associated with the cancer, elimination of a tumor associated withthe cancer, prevention of metastatic cancer, prevention of the cancerand stimulation of effector cell immunity against the cancer.

[0162] A therapeutic composition of the present invention which includesa nucleic acid molecule encoding an immunogen from an infectious diseasepathogen is advantageous for eliciting an immune response in a mammalthat has infectious diseases responsive to an immune response. Aninfectious disease responsive to an immune response is a disease causedby a pathogen in which the elicitation of an immune response against thepathogen can result in a prophylactic or therapeutic effect aspreviously described herein. Such a method provides a long term,targeted therapy for primary lesions (e.g., granulomas) resulting fromthe propagation of a pathogen. As used herein, the term “lesion” refersto a lesion formed by infection of a mammal with a pathogen. Atherapeutic composition for use in the elicitation of an immune responsein a mammal that has an infectious disease comprises a pathogenantigen-encoding recombinant molecule, alone or in combination with acytokine-encoding recombinant molecule of the present invention,combined with a liposome delivery vehicle. Similar to the mechanismdescribed above for the treatment of cancer, eliciting an immuneresponse in a mammal that has an infectious disease with immunogens fromthe infectious disease pathogens with or without cytokines can result inincreased T cell, natural killer cell, and macrophage cell activity thatovercome the relative lack of immune response to a lesion formed by apathogen. Preferably, expression of the immunogen in a tissue of amammal that has an infectious disease produces a result which includesalleviation of the disease, regression of established lesions associatedwith the disease, alleviation of symptoms of the disease, immunizationagainst the disease and stimulation of effector cell immunity againstthe disease.

[0163] A therapeutic composition of the present invention isparticularly useful for eliciting an immune response in a mammal thathas an infectious diseases caused by pathogens, including, but notlimited to, bacteria (including intracellular bacteria which reside inhost cells), viruses, parasites (including internal parasites), fungi(including pathogenic fungi) and endoparasites. Preferred infectiousdiseases to treat with a therapeutic composition of the presentinvention include chronic infectious diseases, and more preferably,pulmonary infectious diseases, such as tuberculosis. Particularlypreferred infectious diseases to treat with a therapeutic composition ofthe present invention include human immunodeficiency virus (HIV),Mycobacterium tuberculosis, herpesvirus, papillomavirus and Candida.

[0164] In one embodiment, an infectious disease a therapeuticcomposition of the present invention is a viral disease, and preferably,is a viral disease caused by a virus which includes, humanimmunodeficiency virus, and feline immunodeficiency virus.

[0165] A therapeutic composition of the present invention which includesa nucleic acid molecule encoding an immunogen that is an allergen isadvantageous for eliciting an immune response in a mammal that has adisease associated with allergic inflammation. A disease associated withallergic inflammation is a disease in which the elicitation of one typeof immune response (e.g., a Th2-type immune response) against asensitizing agent, such as an allergen, can result in the release ofinflammatory mediators that recruit cells involved in inflammation in amammal, the presence of which can lead to tissue damage and sometimesdeath. The method of the present invention, as described in detail inthe Examples section, elicits a Th1-type response, which, without beingbound by theory, the present inventors believe can have prophylactic ortherapeutic effects such that allergic inflammation is alleviated orreduced. A therapeutic composition for use in the elicitation of animmune response in a mammal that has a disease associated with allergicinflammation comprises an allergen-encoding recombinant molecule, aloneor in combination with a cytokine-encoding recombinant molecule,combined with a liposome delivery vehicle. Similar to the mechanismdescribed above for the treatment of cancer, eliciting an immuneresponse in a mammal that has a disease associated with allergicinflammation with allergens with or without cytokines can result inincreased Th1-type T cell, natural killer cell, and macrophage cellactivity that overcome the harmful effects of a Th2-type immune responseagainst the same allergen. Preferably, expression of the allergen in atissue of a mammal that has a disease associated with allergicinflammation produces a result which includes alleviation of thedisease, alleviation of symptoms of the disease, desensitization againstthe disease and stimulation a protective immune response against thedisease.

[0166] Preferred diseases associated with allergic inflammation whichare preferable to treat using the method and composition of the presentinvention include, allergic airway diseases, allergic rhinitis, allergicconjunctivitis and food allergy.

[0167] The following examples are provided for the purposes ofillustration and are not intended to limit the scope of the presentinvention.

EXAMPLES

[0168] For the following Examples 1-7, the following experimentalmethods and materials were used.

[0169] A. Preparation of Cationic Lipid DNA Complexes (CLDC):

[0170] The cationic liposomes used in the following experiments (unlessotherwise indicated) consisted of DOTAP (1,2dioleoyl-3-trimethylammonium-propane) and cholesterol mixed in a 1:1molar ratio, dried down in round bottom tubes, then rehydrated in 5%dextrose solution (D5W) by heating at 50° C. for 6 hours, as describedpreviously (Solodin et al., 1995, Biochemistry 34:13537-13544,incorporated herein by reference in its entirety). Other lipids (e.g.,DOTMA) were prepared similarly for some experiments as indicated. Thisprocedure results in the formation of liposomes that consists ofmultilamellar vesicles (MLV), which the present inventors have foundgive optimal transfection efficiency as compared to small unilamellarvesicles (SUV). The production of MLVs and related “extruded lipids” isalso described in Liu et al., 1997, Nature Biotech. 15:167-173; andTempleton et al., 1997, Nature Biotech. 15:647-652; both of which areincorporated herein by reference in their entirety. Plasmid DNA (pCR3.1,Invitrogen) was purified from E. coli as described previously, usingmodified alkaline lysis and polyethylene glycol precipitation (Liu etal., 1997, supra). DNA for injection was resuspended in distilled water.Eukaryotic DNA (salmon testis and calf thymus) was purchased from SigmaChemical Company. For many of the experiments reported here, the plasmidDNA did not contain a gene insert (unless otherwise noted), and is thusreferred to as “non-coding” or “empty vector” DNA.

[0171] The cationic lipid DNA complexes (CLDC) used in the experimentsbelow were prepared by gently adding DNA to a solution of lipid in 5%dextrose solution (D5W) at room temperature, then gently pipetting upand down several times to assure proper mixing. The DNA:lipid ratio was1:8 (1.0 ug DNA to 8 nmol lipid). The CLDC were used within 30-60minutes of preparation. To prepare small unilamellar vesicles (SUV) usedin some experiments (as indicated), the CLDC that were formed using MLVliposomes as described above were subjected to sonication for 5 minutes,as described previously (Liu et al., 1997, supra).

[0172] B. Gene Constructs

[0173] For antigen-specific immunization experiments, plasmid-based,eukaryotic expression vectors were utilized to express genes in vivo.Expression vectors (using pCR3.1, Invitrogen) for the cytokine cDNAs(IL-2, IFN*, 1L-12) were all constructed using PCR amplification of RNAprepared from normal mouse spleens as described, for example in Sambrooket al., supra. The *-gal expression construct was provided by Dr. CoriGorman. For immunization with these gene constructs, CLDC containing thedesired gene constructs were injected by tail vein (i.e., intravenousdelivery) or intraperitoneally (as indicated) to deliver a total DNAamount of 5.0 to 10.0 ug DNA.

[0174] For RNA immunization experiments, tumor cells (either B 16 cellsor CT-26 cells; see below) were grown in vitro, followed by extractionof the poly-A enriched RNA using standard procedures (Sambrook, supra).The RNA was resuspended in water and frozen prior to formation ofcomplexes with liposomes. The same lipid:RNA ratios as described abovefor lipid:DNA complexes were used to prepare cationic lipid RNAcomplexes (CLRC).

[0175] When more than one gene was injected simultaneously into the sameanimal, the plasmid DNAs were first mixed and then added to liposomes toform CLDC.

[0176] C. In Vivo Evaluation of Immune Activation

[0177] Mice (3 per group, unless otherwise indicated) were injectedintravenously or intraperitoneally, as indicated in the individualexperiments, once with 100 ul of CLDC (prepared as described above) inD5W. Control mice were injected with 100*1 of D5W only. Three differentstrains of mice were evaluated in these experiments (C57B1/6, BALB/c,ICR), but most of the data was generated using C57B1/6 mice. The totalamount of DNA injected was 10 μg per mouse, unless specified otherwise.At various time points post-injection (as indicated), the spleen andlung tissues were collected, mononuclear cell preparations were made,and the cells were assayed for expression of activation markers orcytokine release (see below).

[0178] D. In Vitro Evaluation of Immune Activation

[0179] Spleen cells obtained from normal (untreated mice) were incubatedin modified Eagles cell culture medium with 10% FBS with either lipidalone, DNA alone, or cationic lipid DNA complexes (CLDC) to assess theeffects on immune activation. The final DNA concentration in theseexperiments was 1.0 μg/ml medium. Cell activation was assessed by flowcytometry and cytokine release was quantitated by ELISA (see below).

[0180] E. Flow Cytometry

[0181] Upregulation of the early activation marker, CD69, which isupregulated on activated T cells, B cells, macrophages and NK cells, wasused to assess early immune cell activation. Single cell suspensionswere prepared from spleens of mice by NH₄Cl lysis procedure (Sambrook,supra), and lung mononuclear cells were prepared from lung tissues bycollagenase digestion. Briefly, lung tissues were digested in 0.02%collagenase at 37° C. for one hour. Lung mononuclear cells were purifiedfrom the digested tissue by Ficoll gradient centrifugation. For eachexperiment, spleen and lung cells were prepared from 3 animals pertreatment group, unless noted otherwise. Cells were analyzed using aBecton-Dickinson FACSCalibur flow cytometer, with analysis gates set byfirst gating on spleen lymphocytes. Between 10,000 and 30,000 gatedevents were analyzed for each cell type. For analysis of cellactivation, 3-color flow cytometric analysis was done, using anti-CD69phycoerythrin (Pharmingen, San Diego, Calif.) to quantitate the numberof CD69 positive cells. Cells were also dual-labeled to evaluate T cells(anti-**TCR antibody (biotin H57.597; Pharmingen) plus antibodies toeither CD4 (FITC RM4-5; Pharmingen) or CD8 (FITC 53-6.7; Pharmingen). Bcells were dual-labeled with anti-B220 (Pharmingen) and anti-IA^(b)(FITC 3F12.35; provided by Dr. John Freed, National Jewish) oranti-IA^(d) (FITC 14.44); NK cells were dual-labeled using anti NK 1.1(biotin PK136; Pharmingen) and anti CD3 (FITC 2C11); macrophages wereevaluated using anti-CR3 (biotin Mac-1; Pharmingen) and FITC anti-IA^(b)or anti-IA^(d). The percentage of double positive cells expressing CD69was determined for each cell type, and the mean (±SD) CD69+ cellsplotted.

[0182] F. Cytotoxicity Assay

[0183] A standard 4-hour ⁵¹Cr-release assay was used to quantitatecytotoxic activity present in freshly isolated lung and spleenmononuclear cells, using YAC-1 cells as targets. Briefly, effector cellsfrom lung or spleen were added in decreasing concentrations to duplicatewells of a Linbro plate, to which was then added 5×10³ target cells thathad been previously labeled for 1 hour with ⁵¹Cr. The plates wereincubated at 37° C. for 4 hours, then supernatants from each well wereharvested and the amount of radioactive Cr present was determined byautomated gamma counter. The percentage specific lysis was calculated asfollows:$\frac{{\left( {{observed}\quad {\,^{51}{Cr}}\quad {release}} \right) - \left( {{spontaneous}\quad {\,^{51}{Cr}}\quad {release}} \right)}\quad}{{\left( {{maximum}\quad {\,^{51}{Cr}}\quad {release}} \right) - \left( {{spontaneous}\quad {\,^{51}{Cr}}\quad {release}} \right)}\quad} \times 100$

[0184] G. NK Cell Depletion In Vivo

[0185] Mice were depleted of NK cells in vivo by a singleintraperitoneal (i.p.) injection of 50 μl rabbit anti-asialoGM1antiserum (Wako BioProducts, Richmond, Va.). Control animals wereinjected with 50 μl non-immune rabbit serum. In other experiments, micewere depleted of NK cells by i.p. injection of a monoclonal antibody toNK cells (PK-136), and control mice were injected with an irrelevant,isotype-matched antibody. It was confirmed that these treatmentseliminated detectable NK cells in spleen and lung (as determined by flowcytometry) and also eliminated cytotoxic activity in spleen cells (datanot shown).

[0186] H. Cytokine Assays

[0187] Cytokine release was measured in spleen cell supernatants aftereither in vivo or in vitro stimulation. For assay of cytokine releaseafter in vivo stimulation, spleen or lung mononuclear cells wereprepared from mice either 6 or 24 hours after i.v. injection, thencultured at a concentration of 5×10⁶ cells/ml for an additional 18 hoursbefore supernatants were harvested. For in vitro stimulation of cytokinerelease, spleen cells were incubated in vitro with DNA, lipid, or DNAplus lipid at a final DNA concentration of 1.0 μg DNA per ml for 18hours, at which time the supernatants were harvested for cytokine assay.Interferon-gamma (IFN*) was assayed using a sandwich ELISA as is knownin the art.

[0188] I. Tumor Challenge Experiments

[0189] The B16 (clone F1O) cells were obtained from Dr. Isiah Fidler (MD Anderson, Houston, Tex.); MCA-205 cells were provided by Dr JackRoutes (National Jewish); CT-26 cells were provided by Dr. NicholasRestifo (National Cancer Institute); 4T1 cells were provided by Dr.Susan Rosenberg). All cell lines were maintained at 37° C. in ModifiedEagles medium supplemented with essential and non-essential amino acids,penicillin and glutamine, and 5% fetal bovine serum, and were treatedperiodically with ciprofloxacin (101g/ml) to maintain mycoplasma-freeconditions. The *-gal transfected CT-26 tumor cell line (known as CL-25)was also provided by Dr. Nicholas Restifo.

[0190] To establish experimental pulmonary metastases, mice (4 pertreatment group) were injected once via the lateral tail vein with2.5×10⁵ tumor cells. Treatment with DNA-lipid complexes was initiated 3days after tumor injection, and was repeated once on day 10 after tumorinjection; control mice were injected i.v. with D5W alone. Mice weresacrificed on day 17 to 20 after tumor injection, and the number oftumor nodules per lung was determined by insufflating lungs with Indiaink solution and manually counting total nodules per lung under a tumordissecting microscope (Wexter et al., 1966, J. Natl. Cancer Inst.36:641-645, incorporated herein by reference in its entirety).

Example 1

[0191] The following experiments a-l and FIGS. 1-12 show thatsystemically administered cationic liposome DNA complexes (CLDC) formedwith non-coding DNA (empty vector) elicit potent immune responses invivo.

[0192] (a) The following experiment shows that intravenous (i.v.)injection of CLDC containing empty vector DNA induces marked activationof 5 different immune effector cell populations in vivo. In thisexperiment, CLDC were prepared which consisted of DOTAP and cholesterolmixed in a 1:1 molar ratio complexed with empty vector plasmid DNA (seeSection A above). C57B1/6 mice were injected intravenously with 100*1 ofCLDC (10 μg empty vector DNA per mouse) in DW5 as described (Section C).24 hours post-injection, spleen cells were harvested from control miceinjected with diluent (D5W), and from mice injected with CLDC. Cellswere labeled with specific antibodies to evaluate CD4+ and CD8+ T cells,NK cells, B cells, and macrophages and with an antibody to CD69 (earlyactivation marker) and analyzed by flow cytometry (Section E). FIG. 1shows the results from CD69/immune effector cell staining with controlmice (open bars) and 3 CLDC-injected mice (black bars). Injection ofCLDC (empty vector) induced pronounced upregulation of CD69 expressionon all relevant immune effector cell populations, and similar resultswere observed as early as 6 hours post-administration (data not shown).These results indicate that systemic administration of CLDC (emptyvector) induces massive and rapid immune activation.

[0193] (b) The following experiment shows that CLDC, but not lipid orDNA alone, induce immune activation in vivo. C57B1/6 mice were injectedintravenously with DNA alone (empty vector; 10 μg), lipid alone(DOTAP:cholesterol), or DNA+ lipid (CLDC-empty vector) as describedabove (Sections A & C) and upregulation of CD69 expression (immuneactivation) on T cells, NK cells was evaluated 24 hours later by flowcytometry (Section E). The data presented in FIG. 2A (CD69+/CD8+ cells)and 2B (CD69+/NK1.1+ cells) clearly illustrate the synergistic immunestimulatory interaction that occurs when DNA and cationic lipids arecomplexed together. Similar results were also obtained for CD4+ T cells,B cells and macrophages (data not shown).

[0194] (c) The following experiment compares the immune activatingpotencies of LPS, poly I/C, and CLDC (empty vector). C57B1/6 mice wereinjected i.v. with 10 μg each of LPS, poly I/C, or CLDC (10 μg DNA) andspleen cells were analyzed for upregulation of CD69 by flow cytometry 24hours later (as described in Sections A, C, and E). FIG. 3 shows thatinjection of CLDC induced substantially greater immune activation thaneither of the classical immune activating stimuli, LPS or poly I/C,indicative of the extreme immune activating potency of CLDC.

[0195] (d) The following experiment shows that even low dose CLDCadministered by the present method induces significant immuneactivation. C57B1/6 mice were injected i.v. with decreasing doses ofCLDC (empty vector), and immune activation (CD69 upregulation on NKcells) was assessed 24 hours later (see Sections A, C, and E). FIG. 4shows that even an extremely low dose of CLDC (100 ng) was capable ofinducing significant immune activation.

[0196] (e) The following experiment demonstrates that bothintraperitoneal and intravenous administration of CLDC induce potentimmune activation. CLDC (empty vector) were administered to C57B1/6 miceeither intravenously (i.v.) or intraperitoneally (i.p.), and immuneactivation (CD69 upregulation) on splenic NK cells was assessed by flowcytometry (See Sections A, C, and E). FIG. 5 shows that administrationof CLDC by either route induced substantial immune activation, althoughthe i.v. route was more potent than the i.p. route.

[0197] (f) The following experiment shows that the immune activationelicited by administration of CLDC according to the present method canbe induced by different lipid formulations. C57B1/6 mice were injectedi.v. with CLDC (empty vector) prepared using liposomes of severaldifferent lipid compositions, but all formulated as MLVs (as describedin Sections A and C). At 24 hours post injection, the degree of immuneactivation (CD69 upregulation) on spleen cells was assessed (Section E).FIG. 6 shows that equivalent immune activation was induced by lipidshaving 3 different chemical compositions, indicating that the immuneactivating properties of CLDC is a general property and is not dependenton any one particular lipid composition.

[0198] (g) The following experiment demonstrates that immune activationby CLDC is independent of the DNA source. It has been previouslyestablished that bacterial DNA is immunostimulatory in mammals, whereasDNA from eukaryotic sources is not (See, for example, Pisetsky et al.,1996, supra; Pisetsky, 1996, supra; Yamamoto, et al., 1994, supra;Roman, et al., 1997, supra; Krieg, 1996, supra; Sun, et al., 1996,supra; Stacey et al., 1996, supra; Sato, et al., 1996, supra; or Ballas,1996, supra). Therefore, the ability of CLDC formulated with eitherbacterial DNA (empty vector plasmid DNA) or eukaryotic DNA from 2different sources (salmon sperm or calf thymus) was evaluated in vivo.C57B1/6 mice were injected i.v. with CLDC containing DNA from one ofthese sources (each formulated to deliver 10 ug DNA per mouse) (SeeSection A & C). Twenty-four hours after i.v. injection of CLDC, thedegree of CD69 upregulation on splenic NK cells was assessed by flowcytometry (Section E). FIG. 7 illustrates that immune activation wasobserved when mice were injected with CLDC comprised of eithereukaryotic or bacterial DNA. Injection of salmon sperm or calf thymusDNA alone did not induce CD69 upregulation (data not shown). Thus, theimmune activating properties of CLDC are surprisingly independent of theDNA source, and immune activation can also be induced by complexes ofcationic lipids and RNA (see Example 7 below).

[0199] (h) The following experiment shows that cytokine release isinduced by CLDC, but not by DNA or lipid alone. Spleen cells wereincubated for 24 hours in vitro with CLDC (empty vector), DNA alone(empty vector), or lipid alone (DOTAP:cholesterol) and the supernatantswere assayed for IFN* (as well as other cytokines, data not shown) (SeeSections D and H). FIG. 8 shows the results of an IFN* ELISA. As wasobserved for CD69 upregulation, cytokine release is also triggered onlyby the CLDC and not by either component alone. Thus, formation of theDNA-lipid complex clearly markedly accentuates any immune stimulatoryproperties that plasmid DNA and lipid alone might possess.

[0200] (i) The following experiment demonstrates that injection of CLDC,but not poly I/C or LPS, induces IFN* production in vivo. C57B1/6 mice(3 per group) were injected i.v. with 10 μg of either CLDC (emptyvector), poly I/C, or LPS (as described in Sections A & C). Six hourslater, spleen cells were harvested and cultured in vitro for anadditional 12 hours. Then, cytokine levels in the supernatants weremeasured (Section H). FIG. 9 shows that the in vivo cytokine response toCLDC injection was clearly different than the response to 2 otherclassical immune activating stimuli (LPS, poly I/C), therebyillustrating a marked difference between CLDC and other so-callednon-specific immune stimulators.

[0201] (j) The following experiment shows that NK cells are the sourceof IFN* production elicited by i.v. CLDC injection. To determine thecell type producing IFN* after injection of CLDC (empty vector), C57B1/6mice were depleted of NK cells using an anti-NK cell antibody (EV/aNK),or were untreated (control), or injected with CLDC and untreated (EV/−)or injected with CLDC and treated with an irrelevant antiserum (EV/NRS)(as described in Section G). The amount of IFN* elaborated by spleen(FIG. 10A) and lung cells (FIG. 10B) 24 hours after injection of CLDCwas quantitated (Section H). This experiment demonstrates that NK cellsare the primary source of IFN* induced by i.v. administration of CLDC.

[0202] (k) The following experiment shows that intravenous injection ofCLDC induces high levels of NK activity in spleen cells. FIG. 11illustrates that spleen cells harvested 24 hours after i.v. injection ofCLDC (empty vector) exhibit high levels of killing of tumor target cells(tumor cell cytotoxicity) (See Section F). To identify the cell typeresponsible for this tumor cell killing activity, C57B1/6 mice weredepleted of NK cells 48 hours prior to injection of CLDC (asialo GM1) orwere treated with an irrelevant antiserum (NRS) or were untreated(control) (as described in Section G). This experiment indicates that NKcells are the primary cell type responsible for the tumor cell killingactivity elicited by injection of CLDC.

[0203] (l) The following experiment demonstrates that intraperitonealinjection of CLDC induces immune activation. Spleen cells were harvestedfrom C57BI/6 mice 24 hours after intraperitoneal injection of 10 μg CLDC(10 μg DNA) complexes encoding either nothing (empty vector; EV) or theIL-2 gene (IL-2), and assayed for CD69 upregulation in both CD8+ andNK1.1+ cells (Sections A, B, C and E). FIG. 12A (CD8+) and FIG. 12B(NK1.1+) shows that intraperitoneal injection of CLDC with either emptyvector or the IL-2 gene induced immune activation, although the effectwas not as great as that induced by i.v. delivery (see FIG. 5). CLDCencoding IL-2 also demonstrated an enhanced immune activation ascompared to CLDC (empty vector).

Example 2

[0204] The following experiments a-d and FIGS. 13-16 demonstrate thatCLDC formed with non-coding DNA (empty vector) exert potent antitumoreffects in vivo when administered according to the method of the presentinvention.

[0205] (a) The following experiment demonstrates that CLDC exert potentantitumor effects when administered to a mammal by the present method.The antitumor efficacy of CLDC (empty vector) was evaluated in 4different murine models of metastatic cancer: MCA-205 (C57B1/6;fibrosarcoma; FIG. 13A); B16 (C57BI/6; melanoma; FIG. 13B); CT26(BALB/c; colon carcinoma; FIG. 13C); and 4T1 (BALB/c; breast cancer;FIG. 13D). In each model, tumors were established in the lungs of mice(4 per group) by i.v. injection of 2.5×10⁵ tumor cells per mouse (asdescribed in Section I). Three days after the tumor cells were injected,treatment with i.v. administration of 100 μl CLDC was administered (10μg empty vector DNA complexed to MLV liposomes as described in SectionsA and C), and repeated once in 7 days. Control mice were injected withdiluent (D5W). Seven days after the second injection (17 days after thetumor cells were first injected), the mice were sacrificed and thenumber of tumor nodules in the lungs determined by manual counting, asdescribed above (Wexter et al., 1966, supra). FIGS. 13A-D illustratesthe potent antitumor activity exerted by systemically administered CLDC,using 4 different tumor models and 2 different strains of mice (C57B1/6and BALB/c).

[0206] (b) The following experiment shows that systemic administrationof CLDC, but not administration of DNA or lipid alone, induces antitumoractivity. C57B1/6 mice (4 per group) with day 3 established MCA-205tumors (Section I) were treated twice with i.v. injections of either MLVliposomes alone, empty vector DNA alone, or CLDC (empty vector) (SeeSections A and I). The number of lung tumor metastases was determined onday 17 post-tumor injection and the results are shown in FIG. 14. Thisexperiment demonstrates that the CLDC, but neither of the 2 constituents(DNA or lipid) alone, induces antitumor activity.

[0207] (c) The following experiment shows that the antitumor activity ofCLDC (empty vector) is independent of the DNA source. To determinewhether the antitumor activity observed with CLDC in experiments (a) and(b) above was only a property of CLDC formulated with bacterial DNA,mice with day 3 established MCA-205 lung metastases were treated withCLDC that were formed using either plasmid (bacterial) DNA, oreukaryotic DNA (from calf thymus or salmon testis). FIG. 15 showsclearly that CLDC formulated with either bacterial or eukaryotic DNAinduced antitumor activity, though the bacterial DNA had slightly morepotent activity.

[0208] (d) The following experiment demonstrates that the type of CLDCadministered significantly influences antitumor activity of thecomposition. Previous investigators have used CLDC formulated as SUV(small unilamellar vesicles) to target systemic gene transfer to thelungs. The present inventors have found that systemic administration ofCLDC formulated as MLV, however, induce much greater antitumor activity,even when only empty vector DNA is administered. FIG. 16 clearlyillustrates this difference. FIG. 16 shows that, where day 3 MCA-205lung metastases were treated with 101g empty vector DNA administeredusing CLDC formulated as either SUVs or MLVs, the MLV formulationsprovided significantly greater antitumor effects.

Example 3

[0209] The following experiment and FIGS. 17A-C show that intravenousinjection of CLDC induces selective gene expression in pulmonarytissues. C57B1/6 mice were injected i.v. with CLDC encoding a reportergene, courteously provided by Dr. Robert Debs (luciferase; panel a), andthe location of gene expression in various organs was determined 24hours later (See Sections A, B and C). As shown in FIG. 17A, luciferasegene expression was almost exclusively confined to pulmonary tissues. InFIGS. 17B and 17C, i.v. injection of CLDC encoding IL-2 or IFN* resultedin efficient intrapulmonary expression of IL-2 and IFN*, as demonstratedby determination of cytokine expression in lung tissues extracted fromthe mice. Injection of non-coding CLDC (EV) was included as anadditional control.

Example 4

[0210] The following experiment and FIGS. 18A-F demonstrates thatadministration of cytokine genes using CLDC delivery improves theantitumor effect over empty vector alone. Using 3 different tumor modelsas described in Example 2 (MCA-205, FIGS. 18A and 18D; CT26, FIGS. 18Band 18E; B16, FIGS. 18C and 18F), we evaluated the antitumor effects ofi.v. delivery of cytokine genes (IL-2, IFN*, and IL-12) using CLDCcontaining plasmid DNA expressing these genes, and compared theantitumor effects to those induced by empty vector DNA (See Sections A,B, C, and I). In both the day 3 treatment models (FIGS. 18A, 18B and18C) and the day 6 treatment models (FIGS. 18D, 18E and 18F), additionof a cytokine gene that stimulates NK cells induced greater antitumoractivity than the empty vector DNA alone, and this additional antitumoreffect was particularly pronounced in the day 6 treatment models. It isbelieved that the added antitumor effect induced by the cytokine genesenhances and depends to a large degree on the initial immune activationinherent to administration of CLDC.

Example 5

[0211] The following experiment and FIGS. 19A and 19B show thatadministration of CLDC having DNA encoding ovalbumin induces strongsystemic antigen-specific immune responses.

[0212] The following experiment shows that intravenous injection of CLDCencoding an antigen gene induces strong systemic antigen-specific immuneresponses and that intravenous (i.v.) DNA immunization is more potentthan intramuscular (i.m.) DNA immunization. C57BI/6 mice (3 per group)were immunized either intramuscularly (IM) with 100 μg DNA encoding theovalbumin (OVA) gene, or intravenously (IV) with 10 μg CLDC encoding theOVA gene (Sections A, B, C). Three weeks later, spleen cells wereharvested and assayed for their ability (i.e., CTL activity) to lyseOVA-expressing target cells (Section F). The results are shown in FIGS.19A and 19B. To detect OVA-specific CTL, lymphocytes from immunized micewere assayed for cytotoxic activity against a control cell line (opencircles) or an OVA-expressing target cell (filled circles). FIG. 19Aillustrates that there was significantly greater killing of theOVA-expressing target cells, indicating that immunization with CLDCencoding an antigen is an efficient means of inducing antigen-specificimmune responses in vivo. FIG. 19B shows that administration ofone-tenth of the amount of DNA using CLDC by intravenous administrationinduces equivalent levels of antigen-specific CTL activity observed withintramuscular injection.

Example 6

[0213] The following experiments a-d and FIGS. 20-23 demonstrate thatthe administration of CLDC having DNA encoding a tumor antigen inducesstrong anti-tumor activity and antigen-specific immune responses invivo.

[0214] (a) The following experiment shows that systemic immunizationwith CLDC encoding a tumor antigen induces strong antitumor activity invivo. BALB/c mice (4 per group) were given 2.5×10 CL-25 tumor cells i.v.to establish pulmonary metastases (Section I). The CL-25 tumor line isderived from the CT26 colon carcinoma cell line and has been modified toexpress the *-gal antigen. Three days after administration of the CL-25tumor cells, mice were treated with 2 i.v. administrations of CLDCencoding either nothing (EV) or the *-gal gene (B-gal), one week apart(Sections A, B, and C). One week after the second treatment, the micewere sacrificed and the antitumor effect was quantitated by counting thenumber of lung tumor nodules. FIG. 20 shows that the number of tumorswas significantly reduced by administration of empty vector CLDC (EV),but was even further reduced by administration of CLDC encoding thespecific tumor antigen, *-gal (B-gal). This experiment illustrates theprinciple that i.v. administration of CLDC encoding a tumor antigen (orantigen(s)) is an effective approach to eliciting immune responsesagainst established tumors.

[0215] (b) The following experiment demonstrates that i.v. administeredCLDC-mediated immunization against a tumor antigen induces effectiveantitumor immunity, whereas intramuscular (IM) or intradermal (ID)immunization does not. Mice (4 per treatment group) with day 3established CL25 lung tumors were treated by intravenous DNAimmunization with *-gal DNA (Sections A, B, C, and I). FIG. 21 showsthat mice treated with intramuscular (B-gal/IM) or intradermal(B-gal/ID) administration of 100 μg B-gal DNA showed no detectableantitumor effect as compared to control mice. By contrast, mice treatedwith *-gal CLDC (B-gal/IV; either 10 μg (10) or 1 μg (1) total DNA permouse), had significantly reduced lung tumor burdens compared to controlmice or to mice treated with i.v. administration of empty vector (EV/IV)CLDC, although i.v. administration of empty vector CLDC had a clearantitumor effect as compared to i.m. or i.d. administration of DNA.Thus, administration of {fraction (1/10)}th or {fraction (1/100)}th theamount of tumor antigen DNA using CLDC by i.v. administration was muchmore effective than conventional DNA immunization approaches.

[0216] (c) The following experiment demonstrates that CLDC-mediatedintravenous immunization with a tumor antigen induces anantigen-specific humoral response in vivo. The relative efficiency ofimmunization via different routes of DNA administration was evaluated inBALB/c mice (4 per group) using plasmid DNA encoding the *-galactosidasegene (*-gal). At 2 week intervals, serum was collected from each mouseand assayed for antibodies against the *-gal protein, using an antibodyELISA assay. Mice immunized by the intradermal and intramuscular routewere injected once with 50 μg *-gal plasmid DNA. Mice immunized once bythe intravenous route and intraperitoneal routes received 101g DNA thatwas complexed to a cationic liposome (CLDC). Control animals were nottreated. The mean *-gal-specific antibody level (at a 1:1000 serumdilution) was determined for each group of mice and plotted for each of4 different time points evaluated. FIG. 22 shows that intravenousadministration of CLDC containing 10 μg DNA elicited a similarantigen-specific humoral immune response to intradermal administrationof 50 μg DNA, and both intravenous and intradermal administrationelicited a more potent humoral immune response than eitherintraperitoneal or intramuscular injection of *-gal DNA.

[0217] (d) The following experiment demonstrates that CLDC-mediatedimmunization with a tumor antigen induces antigen-specific production ofIFN * by spleen cells. As another means of assessing the effectivenessof CLDC-mediated immunization, the release of IFN* (a cytokine withantitumor effects) was quantitated in spleen cells of mice that wereimmunized twice, one week apart, with either empty vector CLDC (EV),IL-2 CLDC (i.e., DNA encoding IL-2), or *-gal CLDC (DNA encoding *-gal)(Sections A, B, C & H). FIG. 23 demonstrates that, mice immunized withthe *-gal CLDC mounted a strong antigen specific immune response whenre-challenged in vitro with the CL25 (*-gal transfected) cell line, asmeasured by IFN* production by splenocytes. In contrast, splenocytesfrom mice immunized with either empty vector CLDC (EV) or IL-2 CLDC(IL-2) produced very little IFN*. These data further substantiate theeffectiveness of antigen-specific immunization using CLDC. It isbelieved that this effectiveness stems in large part from the innateimmune response that is triggered by systemic administration of anyCLDC. This strong induction of innate immune responses undoubtedlyserves as a powerful adjuvant for inducing strong immune responses tothe antigen-encoding DNA.

Example 7

[0218] The following experiments a-b and FIGS. 24 and 25 demonstratethat administration of CLDC having RNA encoding a tumor antigen inducesstrong antitumor immunity and tumor-specific CTL responses in vivo.

[0219] (a) The following experiment shows that CLDC-mediatedimmunization with tumor RNA plus a cytokine induces strong antitumorimmunity. The ability to immunize mice using polyA-enriched RNA fromtumor cells was evaluated by complexing the RNA to a cationic lipid toform cationic lipid RNA complexes (CLRC) (Sections A and B). Theantitumor effects were evaluated in BALB/c mice (4 per treatment group)with day 3 established CT26 lung tumor metastases (Section I). RNA wasprepared from the autologous tumor cells (CT26 RNA) or from anirrelevant control tumor cell line (C57B1/6 RNA), complexed to acationic lipid, then injected i.v. to deliver approximately 501g RNA permouse (Section C). One group of mice was treated with CLDC containingDNA encoding the IL-2 gene alone (IL-2), and a final group was treatedwith CLRC containing both CT26 RNA and DNA encoding the IL-2 gene(CT26+IL-2). The lung tumor burden was quantitated 7 days after thesecond injection of CLDC. FIG. 24 shows that RNA can be effectively usedto immunize mice against a tumor when combined into CLRC and deliveredsystemically, and that this antitumor effect can be enhanced byco-administering the RNA with the DNA encoding IL-2.

[0220] (b) This experiment demonstrates that immunization withtumor-specific RNA induces tumor-specific CTL responses. Mice withestablished CT26 tumors were immunized twice with CLRC containing eitherirrelevant RNA (B16), DNA encoding the IL-2 gene (IL-2), total CT26 RNA(CT26), or total CT26 RNA plus DNA encoding the IL-2 gene (CT26/IL-2)(Sections A, B, and I). One week after the second immunization, spleencells were harvested and assayed for their ability to lyse CT26 targetcells in vitro (Section F). FIG. 25 shows that immunization with eitherCT26 RNA or CT26 RNA plus 1L-2 induced the highest levels of anti-tumorCTL activity. Thus, CLDC-mediated immunization with a broad range(library) of unselected tumor antigens can induce tumor-specificimmunity, and this immunity can be augmented by co-administration of acytokine gene.

Example 8

[0221] The following experiment and FIG. 26 demonstrate thatintraperitoneal administration of CLDC containing DNA encoding IL-2induces a reduction in FeLV viral titer. A cat chronically infected withthe feline leukemia virus (FeLV) was treated with weekly (for 4 weeks),and then twice monthly intraperitoneal injections of 250 μg CLDCprepared (as described above) using plasmid DNA encoding the feline IL-2gene. At various time points after treatment was initiated, blood wascollected and the serum levels of FeLV p27 determined using an ELISA(assays performed by Dr. Ed Hoover, Colorado State University). Over thecourse of 3 months of treatment, the FeLV p27 levels declined by 50%,and the cat's clinical signs improved (e.g., weight gain, increasedhematocrit). In contrast, for 2 months prior to IL-2 CLDC treatment, theFeLV p27 levels had remained relatively constant (data not shown).

Example 9

[0222] The following experiments a-b and FIGS. 27-29 demonstrate thatthe composition and method of the present invention abrogates airwayhyperresponsiveness and reduces airway eosinophil influx in a murinemodel of allergic asthma.

[0223] (a) BALB/c mice (at least 8 per treatment group) were sensitizedto ovalbumin as follows. Briefly, mice were sensitized byintraperitoneal (i.p.) injection of 20 μg ovalbumin (OVA) (Grade V,Sigma Chemical Co., St. Louis, Mo.) together with 20 mg alum (Al(OH)³)(Inject Alum; Pierce, Rockford, Ill.) in 100 *1 PBS (phosphate-bufferedsaline), or with PBS alone. 72 hours before the mice were airwaychallenged with ovalbumin, the mice were treated with intravenousadministration of IFN* CLDC (IFN-g) or empty vector CLDC (EV). Controlsincluded OVA-sensitized mice that were not treated (IPN) as well asuntreated mice that did not receive airway sensitization (IP). Micereceived subsequent OVA aerosol challenge for 20 minutes with a 1%OVA/PBS solution. Airways responsiveness (Penh) following increasingdoses of methacholine was assessed using whole body plethysmography(Buxco, Troy, N.Y.) (asthma is known to increase the sensitivity of theairways to contractile agonists such as methacholine). In this system,an unrestrained spontaneously breathing mouse is placed into the mainchamber of the plethysmograph, and pressure differences between thischamber and a reference chamber are recorded. The resulting box pressuresignal is caused by volume and resultant pressure changes during therespiratory cycle of the animal. From these box pressure signals, thephases of the respiratory cycle, tidal volume, and the enhanced pause(Penh) can be calculated. Penh represents a function of the proportionof maximal expiratory to maximal inspiratory box pressure signals and ofthe timing of expiration. It correlates closely with pulmonaryresistance measured by conventional two-chambered plethysmography inventilated animals. FIG. 27 shows that allergen sensitized andchallenged mice which received intravenous administration of IFN* CLDChad significantly reduced airway hyperresponsiveness to methacholinechallenge (i.e., almost equal to that of control (IP) mice), whereasairways responsiveness remained high in untreated animals (IPN). Animalstreated with empty vector (CLDC) showed reduced hyperresponsiveness tomethacholine at lower methacholine challenge doses. Additionally, bothintravenous administration of IFN* CLDC and empty vector CLDC reducedairway hyperresponsiveness to methacholine significantly better thanadministration of recombinant IFN * protein (data not shown).

[0224] (b) In this experiment, BALB/c mice were sensitized to ovalbuminas described in section (a) above, then treated with CLDC deliveredeither intravenously (IV) or intratracheally (IT). The degree ofeosinophil infiltration into the airways (a measure of airways allergensensitization) was quantitated in bronchoalveolar lavage fluid (BALF).The mean number of eosinophils per ml BALF fluid was plotted for eachgroup of mice (unsensitized control {IP}; sensitized, untreated control{IPN}; and sensitized mice treated with either intratracheal IFN* CLDC,intratracheal EV CLDC, intravenous IFN* CLDC, or intravenous EV CLDC).FIG. 28 demonstrates that treatment with intravenous CLDC (both EV andIFN* CLDC) significantly reduced eosinophil infiltration compared tocontrol (IPN) animals.

Example 10

[0225] The following example demonstrates that spleen and lung cellsfrom mice receiving intravenous, but not intratracheal, administrationof CLDC produce significant amounts of IFN*.

[0226] BALB/c mice were administered CLDC containing 10 μg of DNA eitherintravenously or intratracheally as described in experiments above. 24hours post-administration, IFN* production was measured from isolatedspleen (FIG. 29A) and lung (FIG. 29B) cells of the animals. FIGS. 29Aand 29B show that mice receiving intravenous administration of CLDCproduced significant amounts of IFN* in contrast to mice receivingintratracheal administration of CLDC.

Example 11

[0227] The following example demonstrates that intravenousadministration of CLDC containing DNA encoding IL-2 eradicatesmetastatic lung tumors in a dog.

[0228] A canine patient had a rear limb amputation for osteosarcoma,followed by adjuvant chemotherapy for prevention of tumor metastasis.Osteosarcoma is a highly malignant tumor of dogs that metastasizesreadily to the lungs, even after complete removal (amputation) of theprimary tumor. The median survival time for dogs following amputation is4 months, with death due to tumor metastases. Canine osteosarcoma isthus a highly relevant and useful animal model of osteosarcoma inhumans.

[0229] Six months after this patient underwent amputation and adjuvantchemotherapy, the dog was re-evaluated and metastatic tumors were foundin the lung on thoracic radiographs. The dog was then entered into acancer immunotherapy trial, using intravenously administered CLDCencoding the canine IL-2 gene. The dog was treated weekly for 12 weekswith increasing doses of CLDC, up to a maximum dose of 500 μg (10 μg/kgbody weight). After 6 weeks of treatment, partial tumor regression wasobserved on thoracic radiographs, and by 12 treatments, 90% regressionof lung tumor nodules was observed. Additional treatments have beengiven at once monthly intervals and the dog remains in remission at 1.2years after entering into the study.

[0230] This example demonstrates the potential efficacy of systemicallyadministered CLDC as a cancer treatment in animals in addition to mice.Thus, efficacy was demonstrated in a large, outbred animal (dog) with aspontaneous, highly malignant metastatic tumor (osteosarcoma), withminimal toxicity at the doses employed here.

[0231] In summary, the above-described experiments have demonstrated thefollowing:

[0232] 1. Systemic injection of CLDC containing empty vector(non-coding) plasmid DNA induces intense immune activation, as assessedby upregulation of an early activation marker (CD69), by induction of NKcell cytotoxic activity, increase in NK cell numbers and by induction ofcytokine release in vivo.

[0233] 2. Immune stimulation in vitro or in vivo (at the doses evaluatedhere) is induced by the complex of DNA and cationic lipid (CLDC), andnot by either DNA or lipid alone.

[0234] 3. Immune activation induced by CLDC is quantitatively morepotent than that induced by either LPS (endotoxin) or poly I/C (aclassical inducer of antiviral immune responses). Furthermore, the typeof immune stimulation induced (e.g., the pattern of cytokines induced)also differs qualitatively from that induced by LPS.

[0235] 4. Immune activation by CLDC can be induced by eukaryotic as wellas prokaryotic DNA, indicating that there is some property of the CLDCthat is inherently immune activating, regardless of the source of theDNA.

[0236] 5. Immune activation is induced by complexes of CLDC containingRNA.

[0237] 6. Although any complex of DNA and lipid can conceivably inducesome immune activation, CLDC prepared using MLV liposomes induce themaximal and optimal immune stimulation which induces effective antitumorresponses.

[0238] 7. Systemic administration of tumor antigen genes using CLDC ismore effective than some more conventional routes of DNA immunization(e.g., intramuscular), and equivalent to others (e.g., intradermal athigher doses of DNA), for inducing antigen-specific humoral immunity.Intradermal administration, however, does not provide the anti-tumoreffect observed with systemic administration.

[0239] 8. Systemic administration of one-tenth of the amount of DNAusing CLDC by intravenous administration induces equivalent levels ofantigen-specific CTL activity observed with intramuscular injection.

[0240] 9. Intravenously administered, CLDC-mediated immunization againsta tumor antigen induces effective antitumor immunity, whereasintramuscular (IM) or intradermal (ID) immunization does not.

[0241] 10. Combined administration of an antigen-encoding (i.e.,immunogen-encoding) gene with a cytokine-encoding gene induces greaterimmune responsiveness to the antigen gene, and greater antitumoractivity.

[0242] 11. Systemic i.v. administration of CLDC prepared using MLVliposomes induces preferential transfection of pulmonary tissues.Furthermore, i.v. administration of CLDC encoding certain cytokine genes(e.g., those that stimulate NK cells) induce greater antitumor effects(against established lung tumors) than administration of empty vectorDNA.

[0243] 12. The primary anti-tumor effector cell induced by systemicadministration of CLDC is the NK cell.

[0244] 13. The cytokine response to administration of CLDC ischaracteristic of the response to acute viral infections, and isdominated by release of IFN* from macrophages, NK cells, and other celltypes throughout the body. This pattern of response is ideally suitedfor treatment of cancer, viral infections, and to serve as an adjuvantfor certain types of vaccines.

[0245] 14. Systemic administration of CLDC containing DNA encoding acytokine induces a reduction in viral titer.

[0246] 15. Systemic administration of CLDC containing DNA encoding acytokine abrogates airway hyperresponsiveness and reduces airwayeosinophil influx in an allergic asthma model.

[0247] While various embodiments of the present invention have beendescribed in detail, it is apparent that modifications and adaptationsof those embodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

[0248] The words “comprise,” “comprising,” “include,” “including,” and“includes” when used in this specification and in the following claimsare intended to specify the presence of stated features, integers,components, or steps, but they do not preclude the presence or additionof one or more other features, integers, components, steps, or groupsthereof.

What is claimed is:
 1. A method to elicit an immunogen-specific immuneresponse and a systemic, non-specific immune response in a mammal,comprising administering to said mammal a therapeutic composition by aroute of administration selected from the group consisting ofintravenous and intraperitoneal, said therapeutic compositioncomprising: (a) a liposome delivery vehicle; and, (b) a recombinantnucleic acid molecule comprising an isolated nucleic acid sequenceencoding an immunogen, said nucleic acid sequence being operativelylinked to a transcription control sequence.
 2. The method of claim 1,wherein said route of administration is intravenous.
 3. The method ofclaim 1, wherein said immunogen is selected from the group consisting ofa tumor antigen, an infectious disease pathogen antigen and an allergen.4. The method of claim 1, wherein said therapeutic composition furthercomprises a recombinant nucleic acid molecule having a nucleic acidsequence encoding a cytokine, said nucleic acid sequence beingoperatively linked to a transcription control sequence.
 5. The method ofclaim 4, wherein said nucleic acid sequence encoding said immunogen andsaid nucleic acid sequence encoding said cytokine are in the samerecombinant nucleic acid molecule, said nucleic acid sequences beingoperatively linked to at least one transcription control sequence. 6.The method of claim 4, wherein said nucleic acid sequence encoding saidimmunogen and said nucleic acid sequence encoding said cytokine areoperatively linked to different transcription control sequences.
 7. Themethod of claim 4, wherein said cytokine is selected from the groupconsisting of hematopoietic growth factors, interleukins, interferons,immunoglobulin superfamily molecules, tumor necrosis factor familymolecules and chemokines.
 8. The method of claim 4, wherein saidcytokine is an interleukin.
 9. The method of claim 4, wherein saidcytokine is selected from the group consisting of interleukin-2,interleukin-7, interleukin-12, interleukin-15, interleukin-18, andinterferon-*.
 10. The method of claim 4, wherein said cytokine isselected from the group consisting of interleukin-2, interleukin-12,interleukin-18, and interferon-*.
 11. The method of claim 1, whereinsaid transcription control sequences are selected from the groupconsisting of Rous sarcoma virus (RSV) control sequences,cytomegalovirus (CMV) control sequences, adenovirus control sequencesand Simian virus (SV-40) control sequences.
 12. The method of claim 1,wherein said liposome delivery vehicle comprises lipids selected fromthe group consisting of multilamellar vesicle lipids and extrudedlipids.
 13. The method of claim 1, wherein said liposome deliveryvehicle comprises multilamellar vesicle lipids.
 14. The method of claim1, wherein said liposome delivery vehicle comprises cationic liposomes.15. The method of claim 1, wherein said liposome delivery vehiclecomprises pairs of lipids selected from the group consisting of DOTMAand cholesterol; DOTAP and cholesterol; DOTIM and cholesterol; and DDABand cholesterol.
 16. The method of claim 1, wherein said liposomedelivery vehicle comprises DOTAP and cholesterol.
 17. The method ofclaim 1, wherein expression of said immunogen in a tissue of said mammalelicits said immunogen-specific immune response in said mammal.
 18. Themethod of claim 1, wherein administering said nucleic acid molecule andsaid liposome elicit said systemic, non-specific immune response in saidmammal.
 19. The method of claim 1, wherein said mammal is selected fromthe from the group consisting of humans, dogs, cats, mice, rats, sheep,cattle, horses and pigs.
 20. The method of claim 1, wherein said mammalis a human.
 21. The method of claim 1, wherein said composition has anucleic acid:lipid ratio of from about 1:1 to about 1:64.
 22. The methodof claim 1, wherein said mammal has cancer and wherein said immunogen isa tumor antigen.
 23. The method of claim 22, wherein said therapeuticcomposition further comprises a recombinant nucleic acid molecule havinga nucleic acid sequence encoding a cytokine, said nucleic acid sequencebeing operatively linked to a transcription control sequence.
 24. Themethod of claim 22, wherein said therapeutic composition comprises aplurality of recombinant nucleic acid molecules, each of saidrecombinant nucleic acid molecules comprising a cDNA sequence amplifiedfrom total RNA isolated from an autologous tumor sample, each of saidcDNA sequences encoding a tumor antigen or a fragment thereof and beingoperatively linked to a transcription control sequence.
 25. The methodof claim 22, wherein said therapeutic composition comprises a pluralityof recombinant nucleic acid molecules, each of said recombinant nucleicacid molecules comprising a cDNA sequence amplified from total RNAisolated from a plurality of allogeneic tumor samples of the samehistological tumor type, each of said cDNA sequences encoding a tumorantigen or a fragment thereof and being operatively linked to atranscription control sequence.
 26. The method of claim 22, wherein saidcancer is selected from the group consisting of melanomas, squamous cellcarcinoma, breast cancers, head and neck carcinomas, thyroid carcinomas,soft tissue sarcomas, bone sarcomas, testicular cancers, prostaticcancers, ovarian cancers, bladder cancers, skin cancers, brain cancers,angiosarcomas, hemangiosarcomas, mast cell tumors, primary hepaticcancers, lung cancers, pancreatic cancers, gastrointestinal cancers,renal cell carcinomas, hematopoietic neoplasias, and metastatic cancersthereof.
 27. The method of claim 22, wherein said cancer is selectedfrom the group consisting of a primary lung cancer and a pulmonarymetastatic cancer.
 28. The method of claim 22, wherein said tumorantigen is from a cancer selected from the group consisting ofmelanomas, squamous cell carcinoma, breast cancers, head and neckcarcinomas, thyroid carcinomas, soft tissue sarcomas, bone sarcomas,testicular cancers, prostatic cancers, ovarian cancers, bladder cancers,skin cancers, brain cancers, angiosarcomas, hemangiosarcomas, mast celltumors, primary hepatic cancers, lung cancers, pancreatic cancers,gastrointestinal cancers, renal cell carcinomas, hematopoieticneoplasias and metastatic cancers thereof.
 29. The method of claim 22,wherein said tumor antigen is selected from the group consisting oftumor antigens having epitopes that are recognized by T cells, tumorantigens having epitopes that are recognized by B cells, tumor antigensthat are exclusively expressed by tumor cells, and tumor antigens thatare expressed by tumor cells and by non-tumor cells.
 30. The method ofclaim 22, wherein said expression of said tumor antigen produces aresult selected from the group consisting of alleviation of said cancer,reduction of size of a tumor associated with said cancer, elimination ofa tumor associated with said cancer, prevention of metastatic cancer,prevention of said cancer and stimulation of effector cell immunityagainst said cancer.
 31. The method of claim 22, wherein said expressionof said tumor antigen in a pulmonary tissue by administration of saidcomposition by an intravenous route prevents pulmonary metastatic cancerin said mammal.
 32. The method of claim 1, wherein said mammal has aninfectious disease responsive to an immune response, and wherein saidimmunogen is an infectious disease pathogen antigen.
 33. The method ofclaim 32, wherein said therapeutic composition further comprises arecombinant nucleic acid molecule having a nucleic acid sequenceencoding a cytokine, said nucleic acid sequence being operatively linkedto a transcription control sequence.
 34. The method of claim 32, whereinsaid immunogen is from an infectious disease pathogen selected from thegroup consisting of bacteria, viruses, parasites, and fungi.
 35. Themethod of claim 34, wherein said infectious disease pathogen causes achronic infectious disease in said mammal.
 36. The method of claim 34,wherein said infectious disease pathogen is selected from the groupconsisting of human immunodeficiency virus (HIV), Mycobacteriumtuberculosis, herpesvirus, papillomavirus and Candida.
 37. The method ofclaim 32, wherein said expression of said pathogen antigen in a tissueof said mammal produces a result selected from the group consisting ofalleviation of said disease, regression of established lesionsassociated with said disease, alleviation of symptoms of said disease,immunization against said disease and stimulation of effector cellimmunity against said disease.
 38. The method of claim 32, wherein saidtherapeutic composition comprises a plurality of recombinant nucleicacid molecules, each of said recombinant nucleic acid moleculescomprising a cDNA sequence amplified from total RNA isolated from aninfectious disease pathogen, each of said cDNA sequences encoding animmunogen from said infectious disease pathogen or a fragment thereofand being operatively linked to a transcription control sequence. 39.The method of claim 32, wherein said infectious disease pathogen is avirus.
 40. The method of claim 39, wherein said virus is selected fromthe group consisting of human immunodeficiency virus and felineimmunodeficiency virus.
 41. The method of claim 32, wherein saidinfectious disease is tuberculosis.
 42. The method of claim 41, whereinsaid immunogen is a Mycobacterium tuberculosis antigen.
 43. The methodof claim 41, wherein said immunogen is antigen
 85. 44. The method ofclaim 1, wherein said mammal has a disease associated with allergicinflammation and wherein immunogen is an allergen.
 45. The method ofclaim 44, wherein said therapeutic composition further comprises arecombinant nucleic acid molecule having a nucleic acid sequenceencoding a cytokine, said nucleic acid sequence being operatively linkedto a transcription control sequence.
 46. The method of claim 44, whereinsaid allergen is selected from the group consisting of plant pollens,drugs, foods, venoms, insect excretions, molds, animal fluids, animalhair and animal dander.
 47. The method of claim 44, wherein said diseaseis selected from the group consisting of allergic airway diseases,allergic rhinitis, allergic conjunctivitis, and food allergy.
 48. Themethod of claim 44, wherein said expression of said allergen in a tissueof said mammal produces a result selected from the group consisting ofalleviation of said disease, alleviation of symptoms of said disease,desensitization against said disease, and stimulation of a protectiveimmune response against said disease.
 49. The method of claim 44,wherein said therapeutic composition comprises a plurality ofrecombinant nucleic acid molecules, each of said recombinant nucleicacid molecules comprising a cDNA sequence amplified from total RNAisolated from an allergen, each of said cDNA sequences encoding saidallergen or a fragment thereof and being operatively linked to atranscription control sequence.
 50. A method to elicit a tumorantigen-specific immune response and a systemic, non-specific immuneresponse in a mammal that has cancer, comprising administering to amammal a therapeutic composition by a route of administration selectedfrom the group consisting of intravenous and intraperitonealadministration, said therapeutic composition comprising: (a) a liposomedelivery vehicle; and, (b) total RNA isolated from a tumor sample, saidRNA encoding tumor antigens.
 51. The method of claim 50, wherein saidroute of administration is intravenous.
 52. The method of claim 50,wherein said therapeutic composition further comprises a recombinantnucleic acid molecule having a nucleic acid sequence encoding acytokine, said nucleic acid sequence being operatively linked to atranscription control sequence.
 53. The method of claim 50, wherein saidRNA is enriched for poly-A RNA prior to said administration to saidmammal.
 54. A method to elicit a pathogen-antigen-specific immuneresponse and a systemic, non-specific immune response in a mammal thathas an infectious disease, comprising administering to a mammal atherapeutic composition by a route of administration selected from thegroup consisting of intravenous and intraperitoneal administration, saidtherapeutic composition comprising: (a) a liposome delivery vehicle;and, (b) total RNA isolated from an infectious disease pathogen, saidRNA encoding pathogen antigens.
 55. The method of claim 54, wherein saidroute of administration is intravenous.
 56. A composition for systemicadministration to a mammal to elicit an immunogen-specific immuneresponse and a systemic, non-specific immune response, comprising: (a) aliposome delivery vehicle; and (b) a recombinant nucleic acid moleculecomprising an isolated nucleic acid sequence encoding an immunogen, saidnucleic acid sequence being operatively linked to a transcriptioncontrol sequence; wherein said composition has a nucleic acid:lipidratio of from about 1:1 to about 1:64.
 57. The method of claim 56,wherein said liposome delivery vehicle comprises lipids selected fromthe group consisting of multilamellar vesicle lipids and extrudedlipids.
 58. The method of claim 56, wherein said composition has anucleic acid:lipid ratio of from about 1:10 to about 1:40.
 59. Thecomposition of claim 56, wherein said liposome comprises multilamellarvesicle lipids.
 60. The composition of claim 56, wherein said liposomedelivery vehicle comprises cationic liposomes.
 61. The composition ofclaim 56, wherein said liposome delivery vehicle comprises pairs oflipids selected from the group consisting of DOTMA and cholesterol;DOTAP and cholesterol; DOTIM and cholesterol; and DDAB and cholesterol.62. The composition of claim 56, wherein said liposome delivery vehiclecomprises DOTAP and cholesterol.
 63. The composition of claim 56,further comprising a pharmaceutically acceptable excipient.
 64. Thecomposition of claim 63, wherein said excipient comprises a non-ionicdiluent.
 65. The composition of claim 64, wherein said excipient is 5percent dextrose in water (D5W).